Cyclic peptides for modulating growth of neo-vessels and their use in therapeutic angiogenesis

ABSTRACT

The present disclosure teaches analogs of human chemokines and methods of using them in the prevention, treatment, and ameliorization of diseases that can benefit from therapeutic angiogenesis. The teachings are generally directed to compositions comprising SDF-1 mimetics, as well as methods that include the use of SDF-1 mimetics to induce neo-vessel formation. The disclosure also teaches articles of manufacture that can be useful in practicing the methods taught herein.

BACKGROUND

1. Field of the Invention

This invention relates generally to analogs of human chemokines andmethods of using them in the prevention, treatment, and ameliorizationof diseases that can benefit from therapeutic angiogenesis.

2. Description of the State-of-the-Art

Therapeutic angiogenesis is a relatively new procedure that isrecognized as a viable treatment strategy for increasing the supply ofblood to a tissue in the treatment of a disease. Angiogenesis isgenerally described as the growth of new blood vessels, and there aremany medical situations in which an increase in blood supply isindicated. In a broad sense, this growth of new blood vessels can bederived from an old blood vessel or from bone marrow-derived cells, suchas endothelial progenitor cells and hematopoietic stem cells.

Examples of such situations include, for example, tissue injuries, suchas burns and wound healing, where an increased blood supply can increasethe rate of healing and reduce the risk of infection; cardiovasculardiseases, where an increased blood supply can assist in the repair ofcardiac tissue; peripheral vascular diseases, where an increased bloodsupply can assist in providing sufficient oxygen and nutrients toextremities; a stroke, where an increased blood supply can reduce therisk of transient ischemic attacks and vascular deficiencies that cancreate damage to brain tissue; diabetes, which often includes peripheralvascular disease, for example; and cancer, where drug treatments can beimproved by inducing angiogenesis in a tumor to facilitate transport ofa drug into the cancerous tissue. Accordingly, the methods taught hereinhave many uses, of which a predominant use includes the treatment ofischemic conditions associated with various diseases.

Ischemia is a condition involving a restricted blood flow to a tissueand is the most common consequence of vessel dysfunction. Ischemicconditions result in a disruption of oxygen and nutrient delivery totissue, as well as the accumulation of waste metabolites in tissue.Cells cannot survive an extended case of severe ischemia but may be ableto adapt to a moderate condition where diffusion to and from a borderingnon-ischemic region is capable of sustaining vital cellular functions.Under these moderate conditions, the secondary functions of affectedtissues may be impaired, and a new metabolic equilibrium may beestablished depending on the level of cross-diffusion and hypoxiapresent in the tissue.

In fact, in tissues that normally have a high metabolic turnover, suchas skeletal and cardiac muscle, even a mild case of ischemia can createserious conditions that include hypoxia, acidosis, and a depressedtissue function that may eventually threaten the viability and functionof the tissue. Ischemic cardiac muscle, for example, is particularlyvulnerable to a “reperfusion injury” from ischemia, because thereperfusion that usually must occur in an ischemic cardiac muscle torestore its function introduces free radicals to the ischemic tissueduring the reoxygenation process. In fact, the reperfusion injury cansometimes cause as much damage as the ischemic condition itself.

The options available to one of skill in the art of preventing andtreating ischemia are currently limited. The administration oflipid/cholesterol-lowering agents, diet, and anti-platelet adherencetherapy (e.g. treatment with aspirin) may help slow the progression ofvessel disease in some instances; but surgery may still be the onlyoption in advanced stages of the disease. Such surgeries can includecoronary artery bypass grafting and percutaneous transluminal coronaryangioplasty. Unfortunately, even surgery may not be an option at times.In some cases, the only treatment option may be limited to activatingendogenous angiogenic or arteriogenic pathways to stimulaterevascularization of the ischemic tissue.

Diseases that include the complications of ischemia remain a largeproblem faced by our society. Cardiovascular disease is responsible forover 17 million deaths worldwide each year, and coronary heart diseaseis the biggest contributor. Coronary artery disease is a contributorthat, alone, is responsible for over 550,000 deaths each year in theUnited States. Peripheral vascular diseases create ischemic conditionsthat result in limb amputations for over 150,000 patients each year, andthese patients have a subsequent mortality rate of about 40% within twoyears of amputation.

Our society can benefit significantly from the introduction oftherapeutic methods that can reduce or eliminate the need for surgicalprocedures. The present invention is based on the discovery that selectanalogs of the chemokines known as stromal cell-derived factor-1 (SDF-1)and interleukin-8 (IL-8) are effective at inducing endothelial celldifferentiation, neo-vessel formation and, furthermore, angiogenesis intissue. Accordingly, those skilled in the art will appreciate the noveland effective methods that are taught herein. The teachings represent avaluable contribution to the field of therapeutic angiogenesis, arelatively new field that has been developed to prevent, treat, andameliorate symptoms of, diseases affecting the circulatory system.

SUMMARY

The embodiments taught herein relate generally to analogs of humanchemokines and methods of using them in the prevention, treatment, andameliorization of diseases that can benefit from therapeuticangiogenesis. The methods include the use of SDF-1 mimetics to inducedifferentiation and survival of endothelial cells which, for example,can induce endothelial cell tube formation and, thus, angiogenesis in atissue of a subject. The disclosure also teaches articles of manufacturethat can be useful in practicing the methods taught herein.

In some embodiments, the methods include inducing neo-vessel formation,wherein the method comprises contacting an endothelial cell with acomposition comprising an SDF-1 mimetic, wherein the compositioncomprises an SDF-1 mimetic having the following structure:

(SEQ ID NO:2) R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C).

The underlined residues Lys-Trp-Ile-Gln-Glu form a cyclic portion of themimetic. R_(N) is selected from a group consisting of hydrogen,poly(ethylene glycol) or a derivative thereof, a glycosaminoglycan, abiochemical label, and an N-terminal modifier capable of reducing theability of the SDF-1 mimetic to act as a substrate for aminopeptidases;and R_(C) is selected from a group consisting of a hydroxyl group,poly(ethylene glycol) or a derivative thereof, a glycosaminoglycan, abiochemical label, and a C-terminal modifier capable of reducing theability of the SDF-1 mimetic to act as a substrate forcarboxypeptidases.

The linker consists of four amino acids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, whereinXaa₁, Xaa₂, Xaa₃, and Xaa₄ are each independently selected from a groupconsisting of (a) any natural amino acid, provided that the naturalamino acid is not L- or D-Cys, and (b) any non-natural amino acid havingthe following structure:

R_(L) is selected from a group consisting of saturated and unsaturatedaliphatics and heteroaliphatics consisting of 20 or fewer carbon atomsthat are optionally substituted with (i) a hydroxyl, carboxyl, amino,amido, or imino group; (ii) an aromatic group having from 5 to 7 membersin the ring; or (iii) a group having from 0 to 10 carbon atoms andbearing a positive charge; and, the linker comprises at least one aminoacid having a side chain bearing positive charge.

In some embodiments, the inducing of neo-vessel formation can includemobilizing endothelial cells, mobilizing endothelial progenitor cells,mobilizing hematopoietic stem cells, inducing differentiation ofendothelial cells, inducing survival of endothelial cells, inducingretention of endothelial cells, inducing endothelial tube formation, orany combination thereof. In some embodiments, the methods furtherinclude contacting the SDF-1 mimetic with an endothelial progenitorcell, a hematopoietic stem cell, or a combination thereof.

The methods of the present invention can be used to induce angiogenesisin a tissue of a subject, where the angiogenesis is used in theprevention, treatment, or ameliorization of symptoms of a disease. Insome embodiments, the disease can include ischemia, and the angiogenesisis induced to prevent or repair injury to blood vessels to maintain orincrease blood supply to the tissue. In some embodiments, the diseasecan include cardiovascular disease, peripheral vascular disease, renaldiseases, pulmonary dysfunction, or microvascular angiopathies.

In some embodiments, the methods further include transplantingendothelial progenitor cells (EPCs) to the tissue. And, in someembodiments, the methods further include administering an effectiveamount of an agent in a subject to stimulate bone marrow to inducemobilization of EPCs, and then transplanting the EPCs into a tissue inthe subject to induce angiogenesis. The mimetics of the presentinvention can also be used by themselves, for example, as agents tostimulate bone marrow to release EPCs for transplantation. Other agentsthat may be used include, for example, vascular endothelial growthfactor (VEGF), granulocyte colony stimulating factor (G-CSF), andhepatocyte growth factor (HGF).

In some embodiments, the disease includes cancer, the angiogenesis isinduced to treat, or ameliorate the symptoms of, a cancer by creating avascularity within a tumor for transport of an agent into the tumor.And, in some embodiments, the invention also includes articles ofmanufacture that are useful in practicing the methods taught herein,where the article of manufacture can include, for example, an SDF-1mimetic and instructions for administering the mimetic to a subject inneed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an MTT colorimetric assay that demonstratesthe viability of human umbilical vein endothelial cells after treatmentof the cells with SEQ ID NO:3 according to some embodiments of thepresent invention.

FIG. 2 shows the results of an MTT calorimetric assay that demonstratesthe viability of human umbilical vein endothelial cells after treatmentof the cells with SEQ ID NO:5 according to some embodiments of thepresent invention.

FIG. 3 shows the results of an MTT calorimetric assay that demonstratesthe viability of human umbilical vein endothelial cells after treatmentof the cells with SEQ ID NO:9 according to some embodiments of thepresent invention.

FIG. 4 shows the results of an MTT calorimetric assay that demonstratesthe viability of human umbilical vein endothelial cells after treatmentof the cells with SEQ ID NO:13 according to some embodiments of thepresent invention.

FIG. 5 shows the results of an MTT calorimetric assay that demonstratesthe viability of human umbilical vein endothelial cells after treatmentof the cells with SEQ ID NO:24 according to some embodiments of thepresent invention.

FIGS. 6 a through 6 c show that SDF-1 mimetics induce differentiation ofhuman umbilical vein endothelial cells as seen by the formation of tubestructures when tested using a MATRIGEL tube formation assay accordingto some embodiments of the present invention.

FIGS. 7 a through 7 c show that SDF-1 mimetics induceneo-vascularization when tested using a rat aortic ring assay accordingto some embodiments of the present invention.

FIGS. 8 a through 8 d show that SDF-1 mimetics induce blood vesselformation in vivo when tested using a MATRIGEL plug assay according tosome embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the present invention are generally directed towardmethods of using truncated forms of the human chemokine known as stromalcell-derived factor-1 (SDF-1) in the prevention, treatment, andameliorization of symptoms, of diseases in a subject in need that wouldbenefit from vasculogenesis. The term “treatment” can refer to obtainingbeneficial or desired clinical results that can include, but are notlimited to, alleviation of symptoms; diminishment of the extent of adisease; stabilizing a disease condition; delaying or slowing theprogression of a disease; ameliorating or palliating symptoms of adisease; and partial or total remission, regardless of whether theremission is detectable or undetectable. “Treatment” may also refer totherapeutic and prophylactic measures; as well as to prolonging thesurvival of a patient. A subject that is in “need” of a method taughtherein includes subjects that already have a disease as well as those inwhich the onset of a disease may be prevented. For purposes of thepresent invention, the terms “angiogenesis”, “vasculogenesis”, and“neovascularization” can be used interchangeably in some embodiments andrefer to promoting the growth of new blood vessels from endothelialcells; the terms can also include the demodeling of old blood vessels toincrease blood supply to an ischemic tissue.

SDF-1 includes two isoforms: stromal cell-derived factor-1α (SDF-1α) andstromal cell-derived factor-1β (SDF-1β). The human CXC chemokine SDF-1has a total of 67 amino acid residues as shown below in SEQ ID NO:1:

(SEQ ID NO:1)Lys¹-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Cys-Pro-Cys-Arg-Phe-Phe¹⁴-Glu- 15Ser-His-Val-Ala-Arg-Ala-Asn-Val-Lys-His-Leu-Lys-Ile-Leu-Asn- 30Thr-Pro-Asn-Cys-Ala-Leu-Gln-Ile-Val-Ala-Arg-Leu-Lys-Asn-Asn- 45Asn-Arg-Gln-Val-Cys-Ile-Asp-Pro-Lys-Leu⁵⁵-Lys-Trp-Ile-Gln-Glu- 60Tyr-Leu-Glu-Lys-Ala-Leu-Asn⁶⁷ 67

The amino acids are identified in the present application by thefollowing conventional three-letter abbreviations:

Alanine A Ala Leucine L Leu Arginine R Arg Lysine K Lys Asparagine N AsnMethionine M Met Aspartic acid D Asp Phenylalanine F Phe Cysteine C CysProline P Pro Glutamic acid E Glu Serine S Ser Glutamine Q Gln ThreonineT Thr Glycine G Gly Tryptophan W Trp Histidine H His Tyrosine Y TyrIsoleucine I Ile Valine V Val Ornithine O Orn Other Xaa

The single letter identifier is provided for ease of reference. Thethree-letter abbreviations are generally accepted in the peptide art,recommended by the IUPAC-IUB commission in biochemical nomenclature, andare required by WIPO Standard ST.25. Furthermore, the peptide sequencesare taught according to the generally accepted convention of placing theN-terminus on the left and the C-terminus on the right of the sequencelisting as required by WIPO Standard ST.25.

SDF-1 is functionally distinct from other chemokines in that it plays afundamental role in the trafficking, export and homing of bone marrowprogenitor cells, as well as in the regulation of stem cell andangioblast activity. These activities of SDF-1, such as the regulationof hematopoietic stem cells, can be exploited to produce agents that arehighly useful in therapeutic angiogenesis. Hematopoietic stem cells andangioblasts, for example, are fundamental to revascularization,angiogenesis and neovascularization. SDF-1 is the predominant chemokinewith regard to mobilizing hematopoietic stem cells and endothelialprecursor cells, is upregulated in tissues in response to injuries, andis believed to call stem/progenitor cells to the site of injuries topromote repair. Moreover, SDF-1 has also been shown to induceangiogenesis by recruiting and retaining hematopoietic bone marrow cellsclose to angiogenic vessels. These recruited cells secretepro-angiogenic molecules that activate endothelial cells to generate newblood vessels.

The SDF-1 Mimetics

The present invention is based on the discovery that truncated forms ofSDF-1 have been found to be highly effective at inducing endothelialcell differentiation, tube formation, neo-vessel formation, andangiogenesis. These truncated forms are referred to herein as “SDF-1mimetics” and can be synonymously referred to as “SDF-1 analogs.” Infact, the SDF-1 mimetics can be any analogs, homologs, prodrugs,codrugs, metabolites, congeners, variants, salts, and combinationsthereof, of the truncated forms of SDF-1. The mimetics and theirvariants are referenced using identifiers SEQ ID NO:2 through SEQ IDNO:32, and variants a2 through a32, respectively.

The term “variant” refers to modifications to a peptide that allows thepeptide to retain its binding properties, and such modificationsinclude, but are not limited to, conservative substitutions in which oneor more amino acids are substituted for other amino acids; deletion oraddition of amino acids that have minimal influence on the bindingproperties or secondary structure; conjugation of a linker;post-translational modifications such as, for example, the addition offunctional groups. Examples of such post-translational modifications caninclude, but are not limited to, the addition of modifying groupsdescribed below through processes such as, for example, glycosylation,acetylation, phosphorylation, modifications with fatty acids, formationof disulfide bonds between peptides, biotinylation, PEGylation, andcombinations thereof.

The term “conservatively modified variant” refers to a conservativeamino acid substitution, which is an amino acid substituted by an aminoacid of similar charge density, hydrophilicity/hydrophobicity, size,and/or configuration such as, for example, substituting valine forisoleucine. In comparison, a “non-conservatively modified variant”refers to a non-conservative amino acid substitution, which is an aminoacid substituted by an amino acid of differing charge density,hydrophilicity/hydrophobicity, size, and/or configuration such as, forexample, substituting valine for phenyalanine.

The mimetics can act as an agonist or an antagonist of a nativechemokine and can be designed to include a wide variety of modificationsto provide a diagnostic, therapeutic and/or prophylactic effect in thetreatment of a disease or ameliorization of one or more symptoms of adisease in a subject. The term “subject” and “patient” can be usedinterchangeably in the present invention and refer to an animal such asa mammal including, but not limited to, non-primates such as, forexample, a cow, pig, horse, cat, dog, rat and mouse; and primates suchas, for example, a monkey or a human.

The portions of SDF-1 that are linked together to construct the SDF-1mimetics of the present invention are residues 1-14 and residues 55-67.This numbering system is used herein for reference to the residues inthe mimetics and is shown above in the description of SEQ ID NO:1. Inmost embodiments, the present invention is directed to a method ofinducing neo-vessel formation, where the method includes contacting anendothelial cell with a composition comprising an SDF-1 mimetic havingthe following structure:

(SEQ ID NO:2) a2) R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R^(C).

The term “contacting” refers to placing an agent, such as a compound ofthe present invention in contact with a cellular receptor, and thisplacing can occur in vivo, ex vivo, in situ, or in vitro. In someembodiments, the contacting can include adding an SDF-1 mimetic to aliquid medium containing a cell, and the liquid medium may also containa solvent, such as dimethyl sulfoxide (DMSO), to facilitate the uptakeof the mimetic into the cell. In some embodiments, the contacting caninclude administering an SDF-1 mimetic to a subject in need, wherein theadministering can be performed using any method taught herein, such as,for example, direct injection to a target tissue. Without intending tobe bound by any theory or mechanism of action, the cellular receptorsthat can be activated by the mimetic of the present invention includeCXCR4 and CXCR7, or a combination thereof. In these embodiments, themimetics can act as either agonists or antagonists of the nativechemokine SDF-1. Mimetics of IL-8 have also been found to modulateangiogenesis by acting as agonists or antagonists of the cellularreceptors CXCR1 and CXCR2.

The underlined residues Lys-Trp-Ile-Gln-Glu shown in SEQ ID NO:2 form acyclic portion of the mimetic. It is to be appreciated that a widevariety of amino acid substitutions may also be made in the polypeptidesequences. Examples of such substitutions include, but are not limitedto, substituting lysine for glutamic acid, lysine for aspartic acid,ornithine for glutamic acid, and ornithine for aspartic acid. In someembodiments, the residues 55-67 form a stable α-helix moiety.

The mimetics may be prepared using any technique known in the art. Apeptide or polypeptide component of a mimetic may be composed, at leastin part, of a peptide that has been synthesized, purified, and verified.The binding activity of the native chemokine and its mimetics may alsobe assayed and compared, for example, using standard assay methods. Inaddition, the peptides and polypeptides may also be dimerized through adisulfide bridge formed by gentle oxidation of the cysteines using 10%DMSO in water, purified by HPLC, and verified by mass spectrometry.

In some embodiments, the mimetics may be created by either directly orindirectly connecting at least one modifying group to a reactive groupon the mimetic. The term “modifying group” refers to any functionalgroup composing a portion of a mimetic that was either absent in thenative chemokine or that comprises an isolated sequence of less thanfour amino acids. Such sequences are “isolated” in that they arepositioned differently in the mimetic than they were positioned in thenative chemokine. A modifying group can also be a linker, such as alinker for connecting an agent to create a codrug, and linkers aredescribed below. Examples of such reactive groups include, but are notlimited to, an amino group such as the alpha-amino group at theamino-terminus of a peptide; a carboxyl group at the carboxy-terminus ofa peptide; a hydroxyl group such as those present on a tyrosine, serineor threonine residue; or, any other suitable reactive group on an aminoacid side chain.

The group R_(N) as used in the present invention can be a modifyinggroup and is selected from a group consisting of hydrogen, poly(ethyleneglycol) or a derivative thereof, a glycosaminoglycan, a biochemicallabel, and an N-terminal modifier capable of reducing the ability of theSDF-1 mimetic to act as a substrate for aminopeptidases. Likewise, R_(C)can be a modifying group and is selected from a group consisting of ahydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases.

In most embodiments, residues 1-14 and residues 55-67 are linkedtogether by a linker to form the mimetics of the present invention. Insome embodiments, the linker comprises any combination of natural ornon-natural amino acids, (Xaa), wherein the number of amino acids rangesfrom 1 to about 50; from 4 to about 20; from about 4 to about 12; fromabout 2 to about 40; from about 3 to about 30, or any range therein.

In some embodiments, the linker consists of four amino acids,-Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are eachindependently selected from a group consisting of (a) any natural aminoacid, provided that the natural amino acid is not L- or D-Cys, and (b)any non-natural amino acid. Examples of natural amino acids include, butare not limited to, glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tyrosine, aspartic acid, glutamic acid,lysine, arginine, serine, threonine, cysteine, asparagine, proline,tryptophan, histidine and combinations thereof.

In many embodiments, the linker comprises at least one amino acid havinga side chain bearing positive charge. Examples of such amino acidsinclude Lys, Arg, H is, and Orn. In some embodiments, Xaa₁, Xaa₂, Xaa₃,and Xaa₄ can each be independently selected from a group consisting ofGly, L- or D-Lys, L- or D-Arg, L- or D-H is, and L- or D-Orn. In someembodiments, the linker can contain any combination of Gly and Lys, Glyand Arg, Gly and Orn, or Gly and His. In some embodiments, the linkercan contain all Lys, all Arg, all H is, or all Orn.

In some embodiments, the amino acids may be limited to bifunctionalamino acids or trifunctional amino acids. In some embodiments, the aminoacids may be limited to diamines or triamines. In some embodiments, theamino acids may be limited to monocarboxylics or dicarboxylics. In someembodiments, the amino acids may be limited to aliphatics or aromatics.In some embodiments, the amino acids may be limited to amides. It is tobe appreciated that one skilled in the art should recognize that somegroups, subgroups, and individual amino acids may not be used in someembodiments of the present invention.

In some embodiments, the amino acids are non-natural amino acidsrepresented by a formula:

wherein R_(L) may be a substituted, unsubstituted, hetero-,straight-chained, branched, cyclic, saturated or unsaturated aliphaticradical; or a substituted, unsubstituted, or hetero-aromatic radical. Insome embodiments, R_(L) can be substituted, unsubstituted, orhetero-forms of methyl, iso-propyl, sec-butyl, iso-butyl, benzyl, or acombination thereof.

In embodiments where R_(L) is substituted, examples of substituentsinclude, but are not limited to, hydroxyl, carboxyl, amino, imino groupsand combinations thereof. In embodiments where R_(L) is heteroaliphatic,examples of heteroatoms include, but are not limited to, sulfur,phosphorous, oxygen, nitrogen and combinations thereof. In otherembodiments, R_(L) can comprise substituted or unsubstitutedpoly(alkylene glycols), which include, but are not limited to, PEG, PEGderivatives such as mPEG, poly(ethylene oxide), PPG, poly(tetramethyleneglycol), poly(ethylene oxide-co-propylene oxide), or copolymers andcombinations thereof.

In some embodiments, R_(L) can be a substituted or unsubstitutedalkylene comprising C_(n) carbons in the alkylene backbone, wherein n isan integer ranging from 1 to about 20; from about 2 to about 16; fromabout 3 to about 12; from about 4 to about 10; from about 3 to about 8,and any range therein. In these embodiments, the linker can be, forexample, 11-amino-undecanoic acid. In other embodiments, the linkercomprises any combination of four natural or non-natural amino acids. Insome embodiments, the linker is not -(Gly)-₄.

In some embodiments, R_(L) can be selected from a group consisting ofsaturated and unsaturated aliphatics and heteroaliphatics consisting of20 or fewer carbon atoms that are optionally substituted with (i) ahydroxyl, carboxyl, amino, amido, or imino group; (ii) an aromatic grouphaving from 5 to 7 members in the ring; or (iii) a group having from 0to 10 carbon atoms and bearing a positive charge.

In some embodiments, there is no linker. In other embodiments, themimetics are comprised of portions of the human CXC chemokine SDF-1 thatare connected directly to each other through amide bonds. In otherembodiments, the mimetics are comprised of portions of the human CXCchemokine SDF-1 that are connected by disulfide bonds such as, thedisulfide bonds that can form between Cys residues. In otherembodiments, R_(N) can comprise the peptide sequence Glu-Leu-Arg and alinker. In other embodiments, R_(N) can comprise the peptide sequenceGlu-Leu-Arg and a linker, wherein the Glu-Leu-Arg are the last threeresidues in R_(N). In some embodiments, R_(N) can comprise the peptidesequence Glu-Arg-Leu.

One or more modifying groups may be attached to the mimetics at anysuitable reactive group such as, for example, an amino group, a carboxylgroup, or a hydroxyl group using methods known to those skilled in theart. As described above, examples of chemical connections used to attachthe modifying groups include, but are not limited to, ethers, amides,esters, anhydride, orthoester, alkylamine, sulphide, disulphide,carbamate, all-aromatic carbonate, urea bonds, and the like. A modifyinggroup can be connected, for example, to the N-terminus or C-terminus ofa peptide; to a peptidic or peptidomimetic region flanking the coredomain; to a side chain of at least one amino acid residue such as, forexample, an ε-amino group of a lysyl residue, a carboxyl group of anaspartic acid or glutamic acid residue, a hydroxy group of a tyrosyl,serine or threonine residue, or other suitable reactive group on anamino acid side chain; or in-chain as a linker. Examples of chemicalconnections used to attach the modifying groups include, but are notlimited to, ether, amide, ester, anhydride, orthoester, alkylamine,sulphide, disulphide, carbamate, all-aromatic carbonate, urea bonds, andthe like.

In general, a modifying group can include any of the functional groupsdescribed below. The functional group, for example, may also be a“biotinyl structure”, which includes biotinyl groups and analogues andderivatives thereof. Examples of biotinyl structures include, but arenot limited to, iminiobiotinyl structures such as, for example, a2-iminobiotinyl group. The modifications, for example, may control thepharmacokinetic or pharmacodynamic properties of a mimetic withoutsubstantially reducing its bioactive function, or alter in vivostability, bioavailability, or half-life of the mimetic. In someembodiments, the modifications can provide a diagnostic capability suchas, for example, by creating a means of detecting the presence orlocation of a mimetic in vivo or in vitro. Examples of detectablesubstances are described below.

The functional groups of the present invention can be independentlyselected from substituted, unsubstituted, hetero-, straight-chained,branched, cyclic, saturated or unsaturated aliphatic radical; or asubstituted, unsubstituted, or hetero-aromatic radicals. For example, afunctional group can be selected from H; aliphatic hydrocarbon groupssuch as, for example, alkyl, alkenyl, and alkynyl groups; aromaticgroups such as, for example, aryl, aralkyl, aralkenyl, and aralkynylgroups; and, various other groups as defined below.

In some embodiments of the present invention, the aliphatic radicalshave from about 1 to about 50 carbon atoms, from about 2 to about 40carbon atoms, from about 3 to about 30 carbon atoms, from about 4 toabout 20 carbon atoms, from about 5 to about 15 carbon atoms, from about6 to about 10 carbon atoms, and any range therein. In some embodiments,the aromatic radicals have from about 6 to about 180 carbon atoms, fromabout 12 to about 150 carbon atoms, from about 18 to about 120 carbonatoms, from about 24 to about 90 carbon atoms, from about 30 to about 60carbon atoms, and any range therein.

The term “alkyl” can be used interchangeably with the term “alkylene” insome contexts and refers to a straight-chained or branched hydrocarbonchain. Examples of alkyl groups include lower alkyl groups such as, forexample, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,t-butyl or iso-hexyl; upper alkyl groups such as for example, n-heptyl,n-octyl, iso-octyl, nonyl, decyl, and the like; lower alkylene such as,for example, ethylene, propylene, butylenes, butadiene, pentene,n-hexene and iso-hexene; and upper alkylene such as, for example,n-heptene, n-octene, iso-octene, nonene, decene, and the like. Personsof ordinary skill in the art are familiar with numerous straight-chainedand branched alkyl groups, which are within the scope of the presentinvention. In addition, such alkyl groups may also contain varioussubstituents in which one or more hydrogen atoms can be replaced by afunctional group, or the alkyl groups can contain an in-chain functionalgroup.

The term “alkenyl” refers to a straight-chained or branched hydrocarbonchain where at least one of the carbon-carbon linkages is acarbon-carbon double bond. The term “alkynyl” refers to astraight-chained or branched hydrocarbon chain where at least one of thecarbon-carbon linkages is a carbon-carbon triple bond.

The term “aryl” refers to a hydrocarbon ring bearing a system ofconjugated double bonds often comprising at least six π (pi) electrons.Examples of aromatic groups include, but are not limited to, phenyl,pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl,pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, naphthyl,anysyl, toluoyl, xylenyl, and the like. The term “aralkyl” refers to analkyl group substituted with at least one aryl group. Examples ofaralkyls include substituted benzyls such as, for example, phenylmethyl,2-naphthylethyl, 2-(2-pyridyl)propyl, 5-dibenzosuberyl, and the like.The term “aralkenyl” refers to an alkenyl group substituted with atleast one aryl group. Those aryl groups having heteroatoms in the ringstructure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatics can be substituted at one or more ringpositions and can also be part of a polycyclic group. For example, arylgroups can include fused aromatic moieties such as naphthyl,anthracenyl, quinolyl, indolyl, and the like.

The phrase “straight-chained or branched” includes any substituted orunsubstituted acyclic carbon-containing compounds including, but notlimited to, alkanes, alkenes and alkynes. A radical is“straight-chained” when it has less than 0.1 mole percent of sidechainshaving 1 or more carbon atoms. In some embodiments, a radical isstraight-chained if it has less than 0.01 mole percent of suchsidechains. In other embodiments, a radical is straight-chained if ithas less than 0.001 mole percent of such sidechains. A radical is“branched” when it has more than 0.1 mole percent of sidechains having 1or more carbon atoms. In some embodiments, a radical is branched when ithas more than 0.01 mole percent of such sidechains. In otherembodiments, a radical is branched when it has more than 0.001 molepercent of such sidechains.

The terms “radical,” “group,” “functional group,” and “substituent” canbe used interchangeably in some contexts and can be used together tofurther describe a chemical structure. For example, the term “functionalgroup” can refer to a chemical “group” or “radical,” which is a chemicalstructure variable that is in-chain, pendant and/or terminal to thechemical structure. In some embodiments, a straight chain or branchedalkyl has from about 1 to about 20 carbon atoms, from about 2 to about18 carbon atoms, from about 3 to about 17 carbon atoms, from about 5 toabout 15 carbon atoms, from about 2 to about 10 carbon atoms, or anyrange therein. In other embodiments, a cycloalkyl may have a ringstructure containing from about 2 to about 12 carbon atoms, from about 3to about 11 carbon atoms, from about 4 to about 10 carbon atoms, or anyrange therein.

A functional group may comprise a cyclic or polycyclic group. The term“cyclic group” refers to a ring structure that can be substituted,unsubstituted, hetero-, saturated or unsaturated and have from 3 to 24carbon atoms, from 3 to 18 carbon atoms, from 3 to 12 carbon atoms, orany range therein. Examples of cyclic groups include, but are notlimited to, cycloalkyls such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cyclooctyl structures; cycloalkenes; and aromatics. Theterm “polycyclic group” refers to two or more substituted,unsubstituted, hetero-, saturated or unsaturated cyclic rings in whichtwo or more ring carbons are common among two adjoining rings such thatthe rings are “fused rings.” The rings can also be “bridged rings” inthat they are joined through atoms that are not common among theadjoining rings. The rings can be substituted with substituents such asthose described above.

The term “substituted” is used to characterize a chemical structure thathas been modified by the addition of at least one functional group to atleast one position that can be in-chain, pendant, and/or terminal to thechemical structure. The terms “radical,” “group,” “functional group” and“substituent” can be used interchangeably in some contexts to describe achemical that has been added to another chemical to modify itsstructure. In some embodiments, the functional groups can include, butare not limited to, aliphatics, aromatics, and combinations thereof;alkyls, alkenes, alkynes, cyclic structures, heterocyclic structures,and combinations thereof.

In some embodiments, the functional groups can include, but are notlimited to, oxygen-containing groups such as, for example, alcohols,ethers, phenols, and derivatives thereof. Such oxygen-containing groupsinclude, but are not limited to, acetonides, alcohols, alkoxides,bisphenols, carbinols, cresols, diols, enols, enolates, epoxides,ethers, glycols, hydroperoxides, peroxides, phenols, phenolates,phenoxides, pinacols, trioxides, and ynols.

In some embodiments, the functional groups can include, but are notlimited to, oxygen-containing groups such as, for example, aldehydes,ketones, quinones and derivatives thereof. Such oxygen-containing groupsinclude, but are not limited to, acetals, acyloins, aldehydes, carbonylcompounds, diosphenols, dypnones, hemiacetals, hemiketals, ketals,ketenes, keto compounds, ketones, quinhydrones, quinomethanes, quinines,and combinations thereof.

In some embodiments, the functional groups can include, but are notlimited to, oxygen-containing groups such as, for example, carboxylicacids and derivatives thereof. Such oxygen-containing groups include,but are not limited to, carboxylic acids, oxoacids, sulfonic acids, acidanhydrides, acid thioanhydrides, acyl groups, acyl halides, acylals,anhydrides, carboxylic acids, cyclic acid anhydrides, cyclic anhydrides,esters, fulgides, lactides, lactols, lactones, macrolides, naphthenicacids, ortho acids, ortho esters, oxo carboxylic acids, peroxy acids,and combinations thereof,

In some embodiments, the functional groups can include, but are notlimited to, nitrogen-containing groups containing one nitrogen such as,for example, aldimines, aldoximes, alkoxyamines, amic acids, amides,amines, amine oxides, amine ylides, carbamates, hemiaminals,carbonitriles, carboxamides, isocyamides, cyanates, isocyanates,diisocyanates, cyamides, cyanohydrins, diacylamines, enamines,fulminates, hemiaminals, hydroxamic acids, hydroximic acids,hydroxylamines, imides, imidic acids, imidines, imines, oximes,isoureas, ketenimines, ketimines, ketoximes, lactams, lactims, nitriles,nitro, nitroso, nitrosolic acids, oxime O-ethers, quaternary ammoniumcompounds, quinone imines, quinonoximes, azomethines, ureides,urethanes, and combinations thereof.

In some embodiments, the functional groups can include, but are notlimited to, nitrogen-containing groups containing two or more nitrogenssuch as, for example, aldazines, amide hydrazones, amide oximes,amidines, amidrazones, aminals, amine imides, amine imines, isodiazenes,azans, azides, azo imides, azines, azo compounds, azomethine imides,azoxy compounds, carbodiimides, carboxamidines, diamidides, diazocompounds, diazoamino compounds, diazoates, diazooxides, formamidinedisulfides, formazans, hydrazides, hydrazide hydrazones, hydrazideimides, hydrazidines, hydrazines, hydrazo compounds, hydrazones,ketazines, nitramines, nitrile imines, nitrimines, nitrolic acids,nitrosamides, nitrosamines, nitrosimines, ortho amides, semicarbazones,semioxamazones, triazanes, triazenes, and combinations thereof.

In some embodiments, the functional groups can include, but are notlimited to, sulfur-containing groups such as thio, thiol, thioether,sulfonyl, sulfido, sulfinamides, sulfilimines, sulfimines, sulfimides,sulfinamidines, sulfines, sulfinic acids, sulfinic anhydrides,sulfinylamines, sulfonamides, sulfones, sulfonediimines, sulfonic acids,sulfonic anhydrides, sulfoxides, sulfoximides;

In some embodiments, the functional groups can include, but are notlimited to, silyl groups, halogens, selenoethers, trifluoromethyls,thio-derivatives of urethanes where at least one oxygen atom is replacedby a sulfur atom; phosphoryls, phosphonates, phosphinates; andethyleneically unsaturated groups such as, for example, allyl, acryloyland methacrylol, and maleate and maleimido; and combinations thereof.

Examples of heteroatoms of the hetero-radicals include, but are notlimited to, sulfur, phosphorous, oxygen, nitrogen and combinationsthereof. Examples of heterocyclic groups include, but are not limitedto, pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine,piperazine, and morpholine. The heterocyclic ring may be substituted atone or more positions with substituents such as, for example, halogens,alkyls, cycloalkyls, alkenyls, alkynyls, aryls, arylalkyls, otherheterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides,phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, —CF₃,or —CN. Heterocycles may also be bridged or fused to other cyclicgroups. A linker may also link the heterocyclic group to substituentssuch as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,aryls, arylalkyls, heterocycles, hydroxyls, aminos, nitros, thiolsamines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls,silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers,ketones, aldehydes, esters, —CF3, or —CN.

In some embodiments, the modifying groups can include, but are notlimited to, O-modified derivatives including, but not limited to,C-terminal hydroxymethyl benzyl ether, and other C-terminalhydroxymethyl derivatives; N-modified derivatives including, but notlimited to, substituted amides such as alkylamides; hydrazides andcompounds in which a C-terminal phenylalanine residue is replaced with aphenethylamide analogue such as, for example, by replacing Ser-Ile-Phewith Ser-Ile-phenethylamide.

In some embodiments, the functional group may include afluorescein-containing group. Examples of fluorescein-containing groupsinclude, but are not limited to, 5-(and 6-)-carboxyfluoresceinsuccinimidyl ester and fluorescein isothiocyanate. In some embodiments,the modifying group may include a cholyl structure. An example of acholyl derivative is 3-(O-aminoethyl-iso)-cholyl (Aic).

In some embodiments, the functional group may includeN-acetylneuraminyl, trans-4-cotininecarboxyl,2-imino-1-imidazolidineacetyl, (S)-(−)-indoline-2-carboxyl,2-norbornaneacetyl, γ-oxo-5-acenaphthenebutyryl,(−)-2-oxo-4-thiazolidinecarboxyl group, tetrahydro-3-furoyl group,4-morpholinecarbonyl group, 2-thiopheneacetyl group, 2-thiophenesulfonylgroup, diethylene-triaminepentaacetyl group, (O)-methoxyacetyl group,N-acetylneuraminyl group, and combinations thereof. In some embodiments,the functional group may include light scattering groups, magneticgroups, nanogold, other proteins, a solid matrix, radiolabels,carbohydrates, and combinations thereof.

The mimetics can be used as agents in the treatment of disease, asdescribed herein and, in some embodiments, can also be administered withother agents that are biobeneficial, bioactive, and/or diagnostic. Theseother agents may also be connected to the mimetics as a functionalgroup. A “bioactive agent,” for example, can be connected to a mimeticto provide a therapeutic effect, a prophylactic effect, both atherapeutic and a prophylactic effect, or other biologically activeeffect. A “biobeneficial agent” can also be connected to a mimetic toprovide a biological benefit within a subject. In one example, abiobeneficial agent can be non-inflammatory, such as, for example, byacting as a biomimic to passively avoid attracting monocytes andneutrophils, which leads to the cascade of events creating inflammation.

A “diagnostic agent” is a type of bioactive agent that can be used, forexample, in diagnosing the presence, nature, or extent of a disease ormedical condition in a subject. In one embodiment, a diagnostic agentcan be any agent that may be used in connection with methods for imagingan internal region of a patient and/or diagnosing the presence orabsence of a disease in a patient. Diagnostic agents include, forexample, contrast agents for use in connection with ultrasound imaging,magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR),computed tomography (CT), electron spin resonance (ESR), nuclear medicalimaging, optical imaging, elastography, radiofrequency (RF) andmicrowave laser. Diagnostic agents may also include any other agentsuseful in facilitating diagnosis of a disease or other condition in apatient, whether or not imaging methodology is employed.

In some embodiments, the biobeneficial agents can have a reactive groupthat can be used to connect the agent to a mimetic. Examples of suchreactive groups include, but are not limited to, hydroxyl, carboxyl, andamino groups. In some embodiments, the biobeneficial agents can remainattached to the mimetic or be controllably released from the mimetic.

In some embodiments, the molecular weight of an agent connected to amimetic should be at or below about 40,000 Daltons, or any rangetherein, to ensure elimination of the agent from a subject. In oneembodiment, the molecular weight of the agent ranges from about 300Daltons to about 40,000 Daltons, from about 8,000 Daltons to about30,000 Daltons, from about 10,000 Daltons to about 20,000 Daltons, orany range therein. It is to be appreciated that one skilled in the artshould recognize that some of the groups, subgroups, and individualbiobeneficial agents may not be used in some embodiments of the presentinvention.

Examples of biobeneficial agents include, but are not limited to, manyof the polymers listed above such as, for example,carboxymethylcellulose, poly(alkylene glycols), poly(N-vinylpyrrolidone), poly(acrylamide methyl propane sulfonic acid),poly(styrene sulfonate), sulfonated dextran, polyphosphazenes,poly(orthoesters), poly(tyrosine carbonate), dermatan sulfate,hyaluronic acid, heparin and any derivatives, analogs, homologues,congeners, salts, copolymers and combinations thereof.

Examples of heparin derivatives include, but are not limited to, earthmetal salts of heparin such as, for example, sodium heparin, potassiumheparin, lithium heparin, calcium heparin, magnesium heparin, and lowmolecular weight heparin. Other examples of heparin derivatives include,but are not limited to, heparin sulfate, heparinoids, heparin-basedcompounds and heparin derivatized with hydrophobic materials.

Examples of poly(alkylene glycols) include, but are not limited to, PEG,mPEG, poly(ethylene oxide), poly(propylene glycol)(PPG),poly(tetramethylene glycol), and any derivatives, analogs, homologues,congeners, salts, copolymers and combinations thereof. In someembodiments, the poly(alkylene glycol) is PEG. In some embodiments, thepoly(alkylene glycol) is mPEG. In some embodiments, the poly(alkyleneglycol) is poly(ethylene glycol-co-hydroxybutyrate).

The copolymers that may be used as biobeneficial agents include, but arenot limited to, any derivatives, analogs, homologues, congeners, salts,copolymers and combinations of the foregoing examples of agents.Examples of copolymers that may be used as biobeneficial agents in thepresent invention include, but are not limited to, dermatan sulfate,which is a copolymer of D-glucuronic acid or L-iduronic acid andN-acetyl-D-galactosamine; poly(ethylene oxide-co-propylene oxide);copolymers of PEG and hyaluronic acid; copolymers of PEG and heparin;copolymers of PEG and hirudin; graft copolymers of poly(L-lysine) andPEG; copolymers of PEG and a poly(hydroxyalkanoate) such as, forexample, poly(ethylene glycol-co-hydroxybutyrate); and, any derivatives,analogs, congeners, salts, or combinations thereof. In some embodiments,the copolymer that may be used as a biobeneficial agent can be acopolymer of PEG and hyaluronic acid, a copolymer of PEG and hirudin,and any derivative, analog, congener, salt, copolymer or combinationthereof. In some embodiments, the copolymer that may be used as abiobeneficial agent is a copolymer of PEG and a poly(hydroxyalkanoate)such as, for example, poly(hydroxybutyrate); and any derivative, analog,congener, salt, copolymer or combination thereof.

The bioactive agents can be any moiety capable of contributing to atherapeutic effect, a prophylactic effect, both a therapeutic andprophylactic effect, or other biologically active effect in a subject. Abioactive agent can also have diagnostic properties. The bioactiveagents include, but are not limited to, small molecules, nucleotides,oligonucleotides, polynucleotides, amino acids, oligopeptides,polypeptides, and proteins. Bioactive agents include, but are notlimited to, antiproliferatives, antineoplastics, antimitotics,anti-inflammatories, antiplatelets, anticoagulants, antifibrins,antithrombins, antibiotics, antiallergics, antioxidants, and anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. It is to be appreciatedthat one skilled in the art should recognize that some of the groups,subgroups, and individual bioactive agents may not be used in someembodiments of the present invention.

Antiproliferatives include, for example, actinomycin D, actinomycin IV,actinomycin I1, actinomycin X1, actinomycin C1, and dactinomycin(Cosmegen®, Merck & Co., Inc.). Antineoplastics or antimitotics include,for example, paclitaxel (Taxol®, Bristol-Myers Squibb Co.), docetaxel(Taxotere®, Aventis S. A.), methotrexate, azathioprine, vincristine,vinblastine, fluorouracil, doxorubicin hydrochloride (Adriamycin®,Pfizer, Inc.) and mitomycin (Mutamycin®, Bristol-Myers Squibb Co.), andany prodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof. Antiplatelets,anticoagulants, antifibrin, and antithrombins include, for example,sodium heparin, low molecular weight heparins, heparinoids, hirudin,argatroban, forskolin, vapiprost, prostacyclin and prostacyclinanalogues, dextran, D-phe-pro-arg-chloromethylketone (syntheticantithrombin), dipyridamole, glycoprotein IIb/IIIa platelet membranereceptor antagonist antibody, recombinant hirudin, and thrombininhibitors (Angiomax®, Biogen, Inc.), and any prodrugs, codrugs,metabolites, analogs, homologues, congeners, derivatives, salts andcombinations thereof.

Cytostatic or antiproliferative agents include, for example,angiopeptin, angiotensin converting enzyme inhibitors such as captopril(Capoten® and Capozide®, Bristol-Myers Squibb Co.), cilazapril orlisinopril (Prinivil® and Prinzide®, Merck & Co., Inc.); calcium channelblockers such as nifedipine; colchicines; fibroblast growth factor (FGF)antagonists, fish oil (omega 3-fatty acid); histamine antagonists;lovastatin (Mevacor®, Merck & Co., Inc.); monoclonal antibodiesincluding, but not limited to, antibodies specific for Platelet-DerivedGrowth Factor (PDGF) receptors; nitroprusside; phosphodiesteraseinhibitors; prostaglandin inhibitors; suramin; serotonin blockers;steroids; thioprotease inhibitors; PDGF antagonists including, but notlimited to, triazolopyrimidine; and nitric oxide, and any prodrugs,codrugs, metabolites, analogs, homologues, congeners, derivatives, saltsand combinations thereof. Antiallergic agents include, but are notlimited to, pemirolast potassium (Alamast®, Santen, Inc.), and anyprodrugs, codrugs, metabolites, analogs, homologues, congeners,derivatives, salts and combinations thereof.

Examples of heparin derivatives include, but are not limited to, earthmetal salts of heparin such as, for example, sodium heparin, potassiumheparin, lithium heparin, calcium heparin, magnesium heparin, and lowmolecular weight heparin. Other examples of heparin derivatives include,but are not limited to, heparin sulfate, heparinoids, heparin-basedcompounds and heparin derivatized with hydrophobic materials.

Examples of hyaluronic acid derivates include, but are not limited to,sulfated hyaluronic acid such as, for example, O-sulphated orN-sulphated derivatives; esters of hyaluronic acid wherein the esterscan be aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclicor a combination thereof; crosslinked esters of hyaluronic acid whereinthe crosslinks can be formed with hydroxyl groups of a polysaccharidechain; crosslinked esters of hyaluronic acid wherein the crosslinks canbe formed with polyalcohols that are aliphatic, aromatic, arylaliphatic,cycloaliphatic, heterocyclic, or a combination thereof, hemiesters ofsuccinic acid or heavy metal salts thereof; quaternary ammonium salts ofhyaluronic acid or derivatives such as, for example, the O-sulphated orN-sulphated derivatives.

Other bioactive agents useful in the present invention include, but arenot limited to, free radical scavengers; nitric oxide donors; rapamycin;everolimus; tacrolimus; 40-O-(2-hydroxy)ethyl-rapamycin;40-O-(3-hydroxy)propyl-rapamycin;40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin; tetrazole containingrapamycin analogs such as those described in U.S. Pat. No. 6,329,386;estradiol; clobetasol; idoxifen; tazarotene; alpha-interferon; hostcells such as epithelial cells; genetically engineered epithelial cells;dexamethasone; cytokines; chemokines, chemokine mimetics, chemokinereceptor ligands, and, any prodrugs, codrugs, metabolites, analogs,homologues, congeners, derivatives, salts and combinations thereof.

Free radical scavengers include, but are not limited to,2,2′,6,6′-tetramethyl-1-piperinyloxy, free radical (TEMPO);4-amino-2,2′,6,6′-tetramethyl-1-piperinyloxy, free radical(4-amino-TEMPO); 4-hydroxy-2,2′,6,6′-tetramethyl-piperidene-1-oxy, freeradical (TEMPOL), 2,2′,3,4,5,5′-hexamethyl-3-imidazolinium-1-yloxymethyl sulfate, free radical; 16-doxyl-stearic acid, free radical;superoxide dismutase mimic (SODm) and any analogs, homologues,congeners, derivatives, salts and combinations thereof. Nitric oxidedonors include, but are not limited to, S-nitrosothiols, nitrites,N-oxo-N-nitrosamines, substrates of nitric oxide synthase, diazeniumdiolates such as spermine diazenium diolate and any analogs, homologues,congeners, derivatives, salts and combinations thereof. Chemokinesinclude, but are not limited to, IL-8, IP-10, MIP-1α, RANTES, I-309,MCP-1, CCL28, and SDF-1. Chemokines and chemokine mimetics include, butare not limited to, those taught in U.S. Patent Application PublicationNos. 2002/0156034, 2002/0165123, and 2003/0148940; and U.S. patentapplication Ser. No. 10/243,795; each of which is incorporated byreference herein in its entirety. Chemokine receptor ligands include,but are not limited to, those taught in U.S. Pat. Nos. 6,515,001 and6,693,134; and U.S. Patent Application Publication Nos. 2003/0004136,2003/0045550, 2003/0092674, 2003/0125380, 2005/0059584; each of which ishereby incorporated herein by reference.

Diagnostic agents include, but are not limited to, materials that areradiopaque, radioactive, paramagnetic, fluorescent, lumiscent, anddetectable by ultrasound. In some embodiments, the radiopaque agents arematerials comprising iodine or iodine-derivatives such as, for example,iohexyl and iopamidol. In some embodiments, the radioactive materialsare radioisotopes, which can be detected by tracing radioactiveemissions. Examples of radioactive materials include, but are notlimited to, ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ³⁵S or ³H.

In some embodiments, the paramagnetic agents include, but are notlimited to, gadolinium chelated compounds. Examples of fluorescentagents include, but are not limited to, indocyanine green,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.Examples of agents detectable by ultrasound include, but are not limitedto, perflexane, Albunex® and Optison®. Examples of agents used in PETinclude, but are not limited to, fluorodeoxyglucose, sodium fluoride,methionine, choline, deoxyglucose, butanol, raclopride, spiperone,bromospiperone, carfentanil, and flumazenil. Other examples ofdetectable substances include, but are not limited to, various enzymesand prosthetic groups. Examples of suitable enzymes include, but are notlimited to, horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase. Examples of suitableprosthetic group complexes include, but are not limited to,streptavidin/biotin and avidin/biotin.

Labeled mimetics can be used to assess in vivo pharmacokinetics, as wellas detect the progression of a disease or the propensity of a subject todevelop a disease. For example, chemokine receptors for tissuedistribution can be detected using a labeled mimetic either in vivo orin an in vitro sample derived from a subject. In some embodiments, amimetic may be radioactively labeled with ¹⁴C, either by incorporationof ¹⁴C into the modifying group or one or more amino acid structures inthe mimetic.

A modifying group can be chosen to provide a chelation site for adiagnostic label. In one embodiment, the modifying group can be the Aicderivative of cholic acid, which provides a free amino group. In anotherexample, a tyrosine residue within a mimetic sequence may be substitutedwith radioactive iodotyrosyl. In some embodiments, a mimetic may belabeled with radioactive technetium or iodine. In fact, any isotope ofradioactive iodine may be incorporated to create a diagnostic agent. Insome embodiments, ¹²³I has a half-life of 13.2 hours and can be used forwhole body scintigraphy; ¹²⁴I has a half life of 4 days and can be usedfor PET; ¹²⁵I has a half life of 60 days and can be used for metabolicturnover studies; and, ¹³¹I has a half life of 8 days and can be usedfor whole body counting and delayed low resolution imaging studies.

Aminopeptidases and carboxypeptidases have been found to have importantfunctions in biological activities such as, for example, diabetes,memory and learning, antigen formation, and angiogenesis. The term“aminopeptidase” refers to a multifunctional enzyme that cleavesproteins from the N-terminus. Aminopeptidases can be classified into anumber families such as, for example, the zinc-containing (M1)aminopeptidase family which consists of nine aminopeptidases thatinclude, but are not limited to, placental leucine aminopeptidase(P-LAP), adipocyte-derived leucine aminopeptidase (A-LAP) andleukocyte-derived arginine aminopeptidase (L-RAP).

Modulation of aminopeptidase activity can have many therapeutic andprophylactic applications. In one example, control of the activity ofP-LAP can control the inducement of uterine contractions and treat orprevent disorders such as premature delivery and spontaneous abortion,as well as other disorders associated with water resorption, memory andlearning and glucose metabolism. In another example, control of theactivity of A-LAP can treat disorders associated with antigenproduction, blood pressure and inflammation. In another example, controlof the activity of L-RAP can treat disorders association with antigenformation.

Although both aminopeptidases and carboxypeptidases can terminatebiological activity, the carboxypeptidases clearly predominate in suchterminations. The term “carboxypeptidase” refers to a multifunctionalenzyme that cleaves proteins from the C-terminus. Carboxypeptidases arederived from the zymogens, procarboxypeptidase A and B. Modulation ofcarboxypeptidase activity can have many therapeutic and prophylacticapplications. In one example, control of the activity of thecarboxypeptidases such as kininase II (angiotensin-converting enzyme),carboxypeptidase M, and carboxypeptidase N, can potentially controlhypertensive disorders relating to cardiovascular and kidney disorders.These carboxypeptidases are efficient at cleaving the C-terminalarginine of kinins, which appear to be important regulators ofcardiovascular function; and are likely participants in the actions ofdrugs that affect the heart, kidney, and circulation. The kinins alsohave some role in the regulation of local and systemic hemodynamics;vascular permeability; inflammatory response; activation of neuronalpathways; and movement of electrolytes, water, and metabolic substratesacross epithelia and into other tissues. Accordingly, control ofcarboxypeptidase activity can control the activity of other chemicalssuch as, for example, kinins, and thus can have many therapeuticapplications in the diagnosis and treatment of disease.

In some embodiments, a modification may be introduced at the C-terminusof a peptide, the N-terminus of a peptide, in the region between theC-terminus and N-terminus, or a combination thereof. In someembodiments, a modification to the C-terminus may reduce the ability ofa mimetic to act as a substrate for carboxypeptidases. Examples of suchC-terminal modifiers include, but are not limited to, an amide group, anethylamide group and various non-natural amino acids such as, forexample, D-amino acids and P-alanine. In another embodiment, amodification of a C-terminus may be accompanied by a modification to theN-terminus to reduce the ability of a mimetic to act as a substrate foraminopeptidases. Examples of such N-terminus modifiers include, but arenot limited to acyl, alkyl, aryl, arylalkyl, hydroxyalkyl, alkanoylgroups, alkanoics, diacids, and other modifiers having a carboxylfunctional group. In another embodiment, the modification to anN-terminus can be deamidation.

In another embodiment, a mimetic may be prepared in a “prodrug” form,wherein the mimetic begins acting upon its metabolism in vivo, in whichthe mimetic can become, for example, an agonist or an antagonist. One ofskill can use any known method of preparing such a prodrug and,accordingly, can also prepare “codrug” forms that can carry and deliveranother agent upon metabolism of the codrug in vivo. Examples of suchagents include the bioactive agents, biobeneficial agents, diagnosticagents, and other peptidomimetics, such as the SDF-1 mimetic taughtherein, or IL-8 mimetics. In some embodiments, the agent can ofteninclude a glycosaminoglycan such as for example, heparin, hirudin,hyaluronic acid, and any prodrugs, codrugs, metabolites, analogs,homologues, congeners, derivatives, salts and combinations thereof. Inother embodiments, the agent can be PEG or a derivative thereof.

Phospholipids are an example of another agent that can be administeredwith the mimetics of the present invention. Phospholipids can, forexample, induce regression of fatty plaques or atheromas.Phosphatidylcholine, or lecithin, is an example of a phospholipid thatis a major constituent of cell membranes and important for normalcellular membrane composition and repair. An important characteristic ofphosphatidycholine is that it can reduce plaque depositions on arterialwalls, as well as reduce homocysteine and cholesterol levels.Homocysteine often causes the initial lesions on arterial walls thatallow LDL and fibrinogen to accumulate and eventually obstruct bloodflow. Even if cholesterol and triglyceride levels are not significantlyelevated, homocysteine alone can create an atherosclerosis andthrombosis. Studies in lab animals have shown that phosphatidylcholinecan increase life span by up to 36% in some cases. Phosphatidylcholinecan be combined with chelation therapy, for example, to improve vascularhealth. An example of chelation therapy includes intravenous infusion ofvitamins, magnesium, saline solution and an amino acid, ethylenediaminetriacetate (EDTA).

In some embodiments, the agent can comprise a phospholipid such as, forexample, phosphatidylcholine. In some embodiments, the phospholipids canbe conjugated to any functional group on a mimetic such that the mimeticand phospholipid serve as a codrug. In these embodiments, thephospholipids can be connected to an amino functional group, such as forexample the N-terminus of a mimetic. In these embodiments, for example,the mimetics can be administered with phosphatidylcholine prior toreperfusion of tissue to improve the results of the reperfusion. In someembodiments, the mimetics of the present invention can be administeredwith phosphatidylcholine, chelation therapy, or a combination thereof.In some embodiments, an effective amount of phosphatidylcholine is adaily administration that ranges from about 10 mg/kg to about 1000mg/kg, from about 20 mg/kg to about 800 mg/kg, from about 30 mg/kg toabout 600 mg/kg, from about 40 mg/kg to about 400 mg/kg, from about 40mg/kg to about 200 mg/kg, from about 50 mg/kg to about 100 mg/kg, or anyrange therein.

In most embodiments, the methods of the present invention can be used toprevent, treat, or ameliorate symptoms of, ischemia such as, forexample, by inducing angiogenesis. In some embodiments, the phrase“inducing angiogenesis” can refer to one or a combination of thefollowing four steps: endothelial cell (1) migration; (2)differentiation; (2) survival; and (4) retention. The principal cellsinvolved in angiogenesis are endothelial cells, which line all bloodvessels and constitute virtually the entirety of capillaries. To achievenew blood vessel formation, endothelial cells must first escape fromtheir stable location by breaking through a basement membrane. Once thisis achieved, the endothelial cells migrate toward an angiogenicstimulus, such as might be released from tumor cells, activatedlymphocytes, or wound-associated macrophages. Behind this migratingfront, the endothelial cells proliferate to provide the necessary numberof cells for making a new vessel. Subsequent to this proliferation, thenew outgrowth of endothelial cells needs to reorganize into a patent,three-dimensional, tubular structure.

In some embodiments, the phrase “inducing angiogenesis” can refer tomobilizing endothelial progenitor cells, mobilizing endothelial cells,mobilizing hematopoietic stem cells, inducing differentiation ofendothelial cells, inducing survival of endothelial cells, inducingretention of endothelial cells, inducing endothelial tube formation, orany combination thereof. In some embodiments, the phrase “inducingangiogenesis” can refer to disruption of the basement membrane, cellmigration, cell proliferation, endothelial tube formation, or anycombination of these processes. The occurrence of each of theseprocesses can be verified in vitro, and several in vivo assays can alsobe used to verify the ability of the methods to induce angiogenesis in asubject.

In some embodiments, the methods can induce angiogenesis by recruitingand retaining hematopoietic bone marrow cells close to angiogenicvessels, where these recruited cells secrete pro-angiogenic moleculesthat activate endothelial cells to generate new blood vessels. In someembodiments, the methods can induce endothelial cell migration,proliferation, tube formation, or any combination thereof.

Examples of diseases that can include ischemia include cardiovasculardiseases (CVD), peripheral vascular diseases (PVD), diabetes, andmicrovascular angiopathies. The term “cardiovascular disease” includesdiseases of the heart and of the blood vessels and covers everythingfrom aneurysms to heart attacks to varicose veins. Some types ofcardiovascular diseases create other types of cardiovascular diseases.For example, since a disturbance in SDF-1 signaling may contribute tofunctional impairment of endothelial cell development in patients havinga coronary artery disease (CAD), the use of the mimetics to stimulateSDF-1 signaling May be beneficial in patients with CAD. In someembodiments, stimulating SDF-1 signaling might improve the function ofEPCs and enhance an otherwise impaired neovascularization capacity.

Diseases of the heart include coronary artery disease (CAD), coronaryheart disease, and cardiomyopathy. Coronary artery disease is acondition where blood flow to the heart muscle is impeded due to anobstruction of a coronary artery due to, for example, atherosclerosis.Sclerotic regions of arteries can be opened using scaffolds calledstents, but this procedure can also result in further obstructions dueto restenosis. Coronary heart disease is a term that includes CAD, aswell as the damage caused by the ischemia resulting from the CAD. Thus,coronary heart disease is a manifestation of CAD. Cardiomyopathy is acondition that includes all diseases of the heart muscle, includingischemia of the muscle tissue. Accordingly, the methods of the presentinvention are effective in preventing, treating, and ameliorating thesymptoms of, cardiovascular diseases through the inducement ofangiogenesis.

Diseases of the blood vessels include diseases of the arteries, veins,capillaries, and even lymphatics. Examples of these diseases includearteriosclerosis, atherosclerosis, high blood pressure, stroke,aneurysm, peripheral vascular disease and claudication, vasculitis,thrombosis, varicose veins, and lymphedema. Accordingly, the methods ofthe present invention are effective in preventing, treating, andameliorating the symptoms of, blood vessel diseases through theinducement of angiogenesis. Atherosclerosis is a major cause ofcardiovascular disease, myocardial infarctions, transischemic attacks,stroke, and peripheral vascular disease. Atherosclerosis can result in anarrowing or stenosis, an impeding of blood flow, a loss ofcontractility, hypertension, a formation of aneurysms, and a rupture ofaneurysms, to name a few. Small blood vessels are particularlysusceptible to atherosclerotic narrowing. The narrowing reduces theblood supply to tissue and can result in loss of function, and in somecases, even gangrene. An example of another disease that may be treatedusing the methods taught herein is ischemic bowel disease, which is adisease resulting from a lack of blood flow to bowel tissue. If thenarrowing is to the coronary arteries, of course, the loss of blood flowmay also result in death.

The methods taught herein can be effective at preventing, treating, orameliorating the symptoms of, a peripheral vascular disease. Peripheralvascular disease (PVD) encompasses arterial and venous disease statesthat affect the peripheral circulation, including peripheral arterialdisease (PAD), and these diseases involve the narrowing or obstructionof blood vessels supplying the extremities. PVD is a commonmanifestation of atherosclerosis and most often affects the bloodvessels of the legs. Intermittent claudication is an example of a PAD,in which the blood supply to one or more limbs has been reduced to thepoint at which exercise cannot be sustained without a rapid onset ofcramping and pain; and critical leg ischemia, in which the blood supplyis no longer sufficient to completely support the metabolic needs ofeven a resting limb. Varicose and spider veins are examples ofperipheral vein diseases.

In some embodiments, the methods taught herein can be used to prevent,treat, or ameliorate the symptoms of, a microvascular angiopathy.Microvascular angiopathies are common to many diseases and includeinjuries to small blood vessels to the extent that the tissue suppliedby the blood vessels becomes dysfunctional. The injuries often includeendothelial cell damage or death, and the presence of a coagulation or athrombosis. A type of microvascular angiopathies is thromboticmicroangiopathies (TMA), and the most common cause of TMA is hemolyticuremic syndrome (HUS)—a disease that manifests in renal failure. TMA canalso occur as a complication of pregnancy (eclampsia), with malignanthypertension following irradiation of kidneys, after a transplantationprocedure, with chemotherapies, and with certain infections such as HIV,to name a few.

In some embodiments, the methods taught herein can also be used toprevent, treat, or ameliorate the symptoms of, a renal disease. Specifickidney diseases that can be treated include acute renal failure, HUS,focal glomerulosclerosis, amyloidosis, glomerulonephritis, diabetes,systemic lupus erythematosus (SLE), and chronic hypoxia or atrophy.Diabetes, in particular, is a chronic disease that affects over 5.5million Americans, and over 600,000 new cases of diabetes are diagnosedeach year, leading to over 34,000 deaths. Diabetes targets certaintissues that are vulnerable to the effects of chronically high bloodsugar levels, and these tissues include blood vessels and result inischemia. An example of a type of vascular disease suffered by manydiabetics is lower extremity arterial disease (LEAD), which isidentified by intermittent claudication and/or the absence of peripheralpulses in the lower legs and feet. LEAD, for example, can lead to anincreased mortality in the diabetic, particularly if foot ulcerations,infection, or gangrene occur.

In some embodiments, the methods taught herein can also be used toprevent, treat, or ameliorate the symptoms of, a pulmonary dysfunctionthat can result from injuries to the endothelium of the lungs. Hypoxia,for example, is a pulmonary condition that can result from suchinjuries. These injuries can result from ischemia, for example, as wellas from an immune response, a toxin, or an infection. Other causes ofsuch pulmonary injuries can include hypertension, acute respiratorydistress syndrome and toxic alveolar injury, the latter of which can becreated through smoke inhalation, pneumonia, and pulmonary emboli.

The methods taught herein can be used in treating or ameliorating thesymptoms of cancer. Some cancer treatment suffer from the inability totransport a chemotherapy agent, for example, into a tumor. In someembodiments of the present invention, the angiogenesis is induced tocreate a vascularity within a tumor for transport of an agent, such asan antimitotic, antineoplastic, or antiproliferative, into the tumor.

In some embodiments, the methods taught herein can also be used intreating wounds that were created as a result of any endothelial injury,such as a vascular injury. The injury can be due to an immune systemdisorder including vasculitis, allergic reactions, and autoimmunediseases.

In some embodiments, the methods taught herein can also be used topreserve or enhance the function of organ allografts, which include, butare not limited to, transplants of the kidney, heart, liver, lung,pancreas, skin, bone, intestine, and xenografts.

Pharmaceutical Compositions

The invention further provides pharmaceutical compositions containingthe mimetics. The pharmaceutical compositions include a mimetic in anamount that is diagnostic, therapeutic and/or prophylactic in thediagnosis, prevention, treatment and amelioration of symptoms ofdisease.

The amount of a mimetic used in the compositions can vary according tofactors such as type of disease, age, sex, and weight of the subject.Dosage regimens may be adjusted to optimize a therapeutic response. Insome embodiments, a single bolus may be administered; several divideddoses may be administered over time; the dose may be proportionallyreduced or increased; or any combination thereof, as indicated by theexigencies of the therapeutic situation and factors known one of skillin the art. It is to be noted that dosage values may vary with theseverity of the condition to be alleviated. Dosage regimens may beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions, and the dosage ranges set forth herein areexemplary only and do not limit the dosage ranges that may be selectedby medical practitioners.

The terms “administration” or “administering” refer to a method ofincorporating a compound into the cells or tissues of a subject, eitherin vivo or ex vivo to diagnose, prevent, treat, or ameliorate a symptomof a disease. In one example, a compound can be administered to asubject in vivo parenterally. In another example, a compound can beadministered to a subject by combining the compound with cell tissuefrom the subject ex vivo for purposes that include, but are not limitedto, cell expansion and mobilization assays. When the compound isincorporated in the subject in combination with one or active agents,the terms “administration” or “administering” can include sequential orconcurrent incorporation of the compound with the other agents such as,for example, any agent described above. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include, but arenot limited to, parenteral such as, for example, intravenous,intradermal, intramuscular, and subcutaneous injection; oral;inhalation; intranasal; transdermal; transmucosal; and rectaladministration.

An “effective amount” of a compound of the invention can be used todescribe a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” refers to anamount that is effective at the dosages and periods of time necessary toachieve a desired therapeutic result and may also refer to an amount ofactive compound, prodrug or pharmaceutical agent that elicits anybiological or medicinal response in a tissue, system, or subject that issought by a researcher, veterinarian, medical doctor or other clinicianthat may be part of a treatment plan leading to a desired effect.

The therapeutically effective amount may need to be administered in anamount sufficient to result in amelioration of one or more symptoms of adisorder, prevention of the advancement of a disorder, or regression ofa disorder. In some embodiments, a therapeutically effective amount canrefer to the amount of a therapeutic agent that improves a subject'scondition by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

A “prophylactically effective amount” refers to an amount that iseffective at the dosages and periods of time necessary to achieve adesired prophylactic result. Typically, a prophylactic dose is used in asubject prior to the onset of a disease, or at an early stage of theonset of a disease, to prevent or inhibit onset of the disease orsymptoms of the disease. A prophylactically effective amount may be lessthan, greater than, or equal to a therapeutically effective amount.

In some embodiments, the administration can be oral. In someembodiments, the administration can be subcutaneous injection. In someembodiments, the administration can be intravenous injection using asterile isotonic aqueous buffer. In some embodiments, the administrationcan include a solubilizing agent and a local anesthetic such aslignocaine to ease discomfort at the site of injection. In someembodiments, the administrations may be parenteral to obtain, forexample, ease and uniformity of administration.

The compounds can be administered in dosage units. The term “dosageunit” refers to discrete, predetermined quantities of a compound thatcan be administered as unitary dosages to a subject. A predeterminedquantity of active compound can be selected to produce a desiredtherapeutic effect and can be administered with a pharmaceuticallyacceptable carrier. The predetermined quantity in each unit dosage candepend on factors that include, but are not limited to, (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcreating and administering such dosage units.

A “pharmaceutically acceptable carrier” is a diluent, adjuvant,excipient, or vehicle with which the mimetic is administered. A carrieris pharmaceutically acceptable after approval by a state or federalregulatory agency or listing in the U.S. Pharmacopeial Convention orother generally recognized sources for use in subjects. Thepharmaceutical carriers include any and all physiologically compatiblesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. Examplesof pharmaceutical carriers include, but are not limited to, sterileliquids, such as water, oils and lipids such as, for example,phospholipids and glycolipids. These sterile liquids include, but arenot limited to, those derived from petroleum, animal, vegetable orsynthetic origin such as, for example, peanut oil, soybean oil, mineraloil, sesame oil, and the like. Water can be a preferred carrier forintravenous administration. Saline solutions, aqueous dextrose andglycerol solutions can also be liquid carriers, particularly forinjectable solutions.

Suitable pharmaceutical excipients include, but are not limited to,starch, sugars, inert polymers, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain minor amounts of wetting agents, emulsifying agents, pHbuffering agents, or a combination thereof. The compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationscan include standard carriers such as, for example, pharmaceuticalgrades mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. See Martin, E. W.Remington's Pharmaceutical Sciences. Supplementary active compounds canalso be incorporated into the compositions.

In some embodiments, the carrier is suitable for parenteraladministration. In some embodiments, the carrier can be suitable forintravenous, intraperitoneal, intramuscular, sublingual or oraladministration. In some embodiments, the pharmaceutically acceptablecarrier may comprise pharmaceutically acceptable salts, such as acidaddition salts. For purposes of the present invention, the term “salt”and “pharmaceutically acceptable salt” can be used interchangeably inmost embodiments. Pharmaceutically acceptable salts are non-toxic at theconcentration in which they are administered and include those saltscontaining sulfate, hydrochloride, phosphate, sulfonate, sulfamate,sulfate, acetate, citrate, lactate, tartrate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate,cyclohexylsulfonate, cyclohexylsulfamate, and quinate. Pharmaceuticallyacceptable salts can be obtained from acids, such as hydrochloric acid,sulfuric acid, phosphoric acid, sulfonic acid, sulfonic acid, sulfamicacid, acetic acid, citric acid, lactic acid, tartaric acid, malonicacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, cyclohexylsulfonic acid, cyclohexylsulfamicacid, and quinic acid. Such salts can be prepared, for example, byreacting the free acid or base form of the product with one or moreequivalents of the desired base or acid in a solvent in which the saltis insoluble, or in water that is later removed using a vacuum. Ionexchange can also be used to prepare desired salts.

Pharmaceutical formulations for parenteral administration may includeliposomes. Liposomes and emulsions are delivery vehicles or carriersthat are especially useful for hydrophobic drugs. Depending onbiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed. Furthermore, one mayadminister the drug in a targeted drug delivery system such as, forexample, in a liposome coated with target-specific antibody. Theliposomes will bind to the target protein and be taken up selectively bythe cell expressing the target protein.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable for a high drug concentration. In some embodiments, the carriercan be a solvent or dispersion medium including, but not limited to,water, ethanol; a polyol such as for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like; and, combinationsthereof. The proper fluidity can be maintained in a variety of ways suchas, for example, using a coating such as lecithin, maintaining arequired particle size in dispersions, and using surfactants.

In some embodiments, isotonic agents can be used such as, for example,sugars; polyalcohols that include, but are not limited to, mannitol,sorbitol, glycerol, and combinations thereof; and sodium chloride.Sustained absorption characteristics can be introduced into thecompositions by including agents that delay absorption such as, forexample, monostearate salts, gelatin, and slow release polymers.Carriers can be used to protect active compounds against rapid release,and such carriers include, but are not limited to, controlled releaseformulations in implants and microencapsulated delivery systems.Biodegradable and biocompatible polymers can be used such as, forexample, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid, polycaprolactone,polyglycolic copolymer (PLG), and the like. Such formulations cangenerally be prepared using methods known to one of skill in the art.

Local administration of the mimetics to a target tissue, particular indiseases that include ischemic tissue, can be used in the methods taughtherein. In some embodiments, the mimetics are administered by injectionsthat can include intramuscular, intravenous, intra-arterial,intracoronary, intramyocardial, intrapericardial, intraperitoneal,subcutaneous, intrathecal, or intracerebrovascular injections.

The compounds may be administered as suspensions such as, for example,oily suspensions for injection. Lipophilic solvents or vehicles include,but are not limited to, fatty oils such as, for example, sesame oil;synthetic fatty acid esters, such as ethyl oleate or triglycerides; andliposomes. Suspensions that can be used for injection may also containsubstances that increase the viscosity of the suspension such as, forexample, sodium carboxymethyl cellulose, sorbitol, or dextran.Optionally, a suspension may contain stabilizers or agents that increasethe solubility of the compounds and allow for preparation of highlyconcentrated solutions.

In one embodiment, a sterile and injectable solution can be prepared byincorporating an effective amount of an active compound in a solventwith any one or any combination of desired additional ingredientsdescribed above, filtering, and then sterilizing the solution. Inanother embodiment, dispersions can be prepared by incorporating anactive compound into a sterile vehicle containing a dispersion mediumand any one or any combination of desired additional ingredientsdescribed above. Sterile powders can be prepared for use in sterile andinjectable solutions by vacuum drying, freeze-drying, or a combinationthereof, to yield a powder that can be comprised of the activeingredient and any desired additional ingredients. Moreover, theadditional ingredients can be from a separately prepared sterile andfiltered solution. In another embodiment, a mimetic may be prepared incombination with one or more additional compounds that enhance thesolubility of the mimetic.

In some embodiments, the compounds can be administered by inhalationthrough an aerosol spray or a nebulizer that may include a suitablepropellant such as, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or acombination thereof. In one example, a dosage unit for a pressurizedaerosol may be delivered through a metering valve. In anotherembodiment, capsules and cartridges of gelatin, for example, may be usedin an inhaler and can be formulated to contain a powderized mix of thecompound with a suitable powder base such as, for example, starch orlactose.

In some embodiments, a therapeutically or prophylactically effectiveamount of a mimetic may range in concentration from about 0.001 nM toabout 0.1 M; from about 0.001 nM to about 0.05 M; from about 0.01 nM toabout 15 μM; from about 0.01 μM to about 10 μM, or any range therein. Insome embodiments, the mimetics may be administered in an amount rangingfrom about 0.001 mg/kg to about 50 mg/kg; from about 0.005 mg/kg toabout 40 mg/kg; from about 0.01 mg/kg to about 30 mg/kg; from about 0.01mg/kg to about 25 mg/kg; from about 0.1 mg/kg to about 20 mg/kg; fromabout 0.2 mg/kg to about 15 mg/kg; from about 0.4 mg/kg to about 12mg/kg; from about 0.15 mg/kg to about 10 mg/kg, or any range therein,wherein a human subject is assumed to average about 70 kg.

The mimetics of the present invention can be administered as adiagnostic, therapeutic or prophylactic agent in a combination therapywith the administering of one or more other agents. The agents of thepresent invention can be administered concomitantly, sequentially, orcyclically to a subject. Cycling therapy involves the administering afirst agent for a predetermined period of time, administering a secondagent for a second predetermined period of time, and repeating thiscycling for any desired purpose such as, for example, to enhance theefficacy of the treatment. The agents of the present invention can alsobe administered concurrently. The term “concurrently” is not limited tothe administration of agents at exactly the same time, but rather meansthat the agents can be administered in a sequence and time interval suchthat the agents can work together to provide additional benefit. Eachagent can be administered separately or together in any appropriate formusing any appropriate means of administering the agent or agents.

Each of the agents described herein can be administered to a subject incombination therapy. In some embodiments, the agents can be administeredat points in time that vary by about 15 minutes, 30 minutes, 1 hour, 2hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 48 hours or 1week in time. In some embodiments, at least one of the agents is animmunomodulatory agent. In some embodiments, the agents can includeantiproliferatives, antineoplastics, antimitotics, anti-inflammatories,antiplatelets, anticoagulants, antifibrins, antithrombins, antibiotics,antiallergics, antioxidants, and any prodrugs, codrugs, metabolites,analogs, homologues, congeners, derivatives, salts and combinationsthereof.

The present invention encompasses sustained release formulations for theadministration of one or more agents. In some embodiments, the sustainedrelease formulations can reduce the dosage and/or frequency of theadministrations of such agents to a subject.

In some embodiments, the methods taught herein include administering anagent, such as an SDF-1 mimetic taught herein, to stimulate bone marrowand mobilize EPCs for transplantation, transplanting EPCs to a tissue inneed of vasculogenesis, and combinations thereof. The transplanting ofcells is a form of cell-based therapy for the damaged tissue, and anagent that can stimulate the bone marrow also enhances the mobilizationof repair cells to the target tissue. The EPCs can be autologous,homologous, or heterologous and still incorporate into active angiogenicsites.

In some embodiments, EPCs can be obtained by expanding a cell populationin an ex vivo culture using human peripheral blood mononuclear cells,using techniques known to those skilled in the art. The cells can beobtained from the mononuclear cell fraction of peripheral blood, humanumbilical cord blood, bone marrow-derived mononuclear cells, andhematopoietic stem cells. After expanding the cells to enrich an activesubpopulation of EPCs, the EPCs can be directly injected, for example,into an ischemic tissue, such as myocardial tissue or tissue of anischemic limb. By combining this cell therapy with the administration ofthe SDF-1 mimetics, one of skill in the art can obtain an increase inblood flow recovery, and a decrease in the loss of tissue function.

In some embodiments, an agent can be administered to stimulate asubject's bone marrow into mobilizing bone marrow-derived EPCs. Suchstimulation is a normal reparative response that has been observed insubjects with an ischemic injury. The agent that stimulates the bonemarrow can include an SDF-1 mimetic of the present invention, and otheragents may be administered alone or in combination with the SDF-1mimetics taught herein. These other agents include a hematopoieticgrowth factor known as granulocyte-colony stimulating factor (G-CSF),which is an agent that one of skill will recognize as beign capable ofmobilizing hematopoietic precursor cells from bone marrow andstimulating endothelial cell migration.

Articles of Manufacture

The present invention provides for articles of manufacture thatencompass finished, packaged and labelled pharmaceutical products. Thearticles of manufacture include the appropriate unit dosage form in anappropriate vessel or container such as, for example, a glass vial orother container that is hermetically sealed. In the case of dosage formssuitable for parenteral administration, the active ingredient, e.g. oneor more agents including a chemokine mimetic, is sterile and suitablefor administration as a particulate-free solution. In other words, theinvention encompasses both parenteral solutions and lyophilized powders,each being sterile, and the latter being suitable for reconstitutionprior to injection. Alternatively, the unit dosage form may be a solidsuitable for oral, transdermal, topical or mucosal delivery.

In some embodiments, the unit dosage form is suitable for intravenous,intramuscular, topical or subcutaneous delivery. Thus, the inventionencompasses solutions, which are preferably sterile and suitable foreach route of delivery. The concentration of agents and amountsdelivered are included as described herein.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. In addition, the articles of manufacture can includeinstructions for use or other information material that can advise theuser such as, for example, a physician, technician or patient, regardinghow to properly administer the composition as a diagnostic,prophylactic, therapeutic, or ameliorative treatment of the disease ofconcern. In some embodiments, instructions can indicate or suggest adosing regimen that includes, but is not limited to, actual doses andmonitoring procedures.

In some embodiments, the instructions can include informational materialindicating that the administering of the compositions can result inadverse reactions including but not limited to allergic reactions suchas, for example, anaphylaxis. The informational material can indicatethat allergic reactions may exhibit only as mild pruritic rashes or maybe severe and include erythroderma, vasculitis, anaphylaxis,Steven-Johnson syndrome, and the like. The informational material shouldindicate that anaphylaxis can be fatal and may occur when any foreignprotein is introduced into the body. The informational material shouldindicate that these allergic reactions can manifest themselves asurticaria or a rash and develop into lethal systemic reactions and canoccur soon after exposure such as, for example, within 10 minutes. Theinformational material can further indicate that an allergic reactionmay cause a subject to experience paresthesia, hypotension, laryngealedema, mental status changes, facial or pharyngeal angioedema, airwayobstruction, bronchospasm, urticaria and pruritus, serum sickness,arthritis, allergic nephritis, glomerulonephritis, temporal arthritis,eosinophilia, or a combination thereof.

In some embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and at least one unit dosage form of an agent comprising amimetic within the packaging material. In some embodiments, the articlesof manufacture may also include instructions for using the compositionas a diagnostic, prophylactic, therapeutic, or ameliorative treatmentfor the disease of concern.

In some embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and a first composition comprising at least one unitdosage form of an agent comprising a mimetic within the packagingmaterial, along with a second composition comprising a second agent suchas, for example, a glycosaminoglycan, phospholipid, poly(alkyleneglycol), any other bioactive agent taught herein, or any prodrugs,codrugs, metabolites, analogs, homologues, congeners, derivatives, saltsand combinations thereof. In some embodiments, the articles ofmanufacture may also include instructions for using the composition as adiagnostic, prophylactic, therapeutic, or ameliorative treatment for thedisease of concern.

EXAMPLES Example 1

Peptides of the invention may be synthesized chemically from theC-terminus to the N-terminus (“reverse sequence”) using the Fmoc/tBustrategy either manually or automatically using a batchwise orcontinuous flow peptide synthesizer.

Reagents and Procedures

Main Solvent: a grade certified, ACS spectroanalyzed,N,N-dimethylformamide (DMF) (Fisher, D131-4). The DMF is treated withactivated molecular sieves, type 4A (BDH, B54005) for at least two weeksand then tested with 2,4-dinitrofluorobenzene (FDNB) (Eastman). Equalvolumes of an FDNB solution (1 mg/ml of FDNB in 95% EtOH) and DMF aremixed and allowed to stand for 30 minutes. The absorbance of the mixtureis then taken at 381 nm over an FDNB blank solution (no DMF), and if theabsorbance is approximately 0.2, then the DMF is suitable for thesynthesis

Deblocking Agent: 20% piperidine (Aldrich, 10, 409-4) in DMF containing0.5% (v/v) triton X100 (Sigma, T-9284).

Activating Agents: 2-(H-benzotriazol-lyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU) (Quantum RichelLeu, R0139);hydroxybenzotriazole (HOBt) (Quantum RichelLeu, R0166-100), each at aconcentration of 0.52 M in DMF; and 4-methylmorpholine (NMM) (Aldrich,M5 655-7) at a concentration of 0.9 M in DMF. In the case of amino acidssensitive to racemization such as, for example, cysteine, a2,4,6-collidine (Aldrich, 14, 238-7) is used at a concentration of 0.78M in a 1/1 (v/v) mixture of DMF/dichloromethane (DCM).

Support Resin: TentaGel R RAM (90 μm) beads are used with a9-fluorenylmethoxycarbonyl (Fmoc) Rink-type linker (Peptides Int'l,RTS-9995-PI) in a column. The synthesis begins using 0.5 g of the resinwith a degree of substitution of 0.21 mmol/g for 0.21(0.5) or 0.101 mmolof peptide.

An Fmoc-L-amino derivative is prepared with protected side-chains. Theside-chains are protected using t-butoxycarbonyl (Boc), t-butyl (tBu),and triphenylmethyl (Trt) groups in a 4 fold excess (Peptides Int'l;Bachem; Novabiochem; Chem-Impex, Inc). The Glu⁶⁰ and Lys⁵⁶ residues areAllyl-protected (Millipore/Perseptive Biosys.).

Initial Amino Loading and Peptide Synthesis Procedure

The synthesis starts from the C-terminus, and the residues are doublecoupled automatically at ambient temperature using a 4-fold excess ofthe residues and the coupling reagents, TBTU and HOBt in DMF, for eachcoupling. Double coupling is used to ensure a high yield of coupling andcan be a second coupling step that follows single coupling.

The synthesis is interrupted after Leu⁵⁵ for lactamization of residuesGlu⁶⁰ and Lys⁵⁶ away from the column. The peptide bound to the supportis cyclized by first removing the lateral allyl groups from Glu⁶⁰ andLys⁵⁶ as described below. The peptide synthesis was then resumed.

Removal of the Allyl Groups

The support-bound peptide is removed from the column and a 3-foldsolution (347 mg) of tetrakis(triphenylphosphine) palladium(0)(Pd(PPh₃)₄) (Sigma-Aldrich, 21, 666-6) and 0.1 mmol of the peptideattached to the resin is dissolved in 5% acetic acid. The peptide isactivated using 2.5% NMM in CHCl₃ at a concentration of 0.14 M under anargon purge. The solution is added to the support-bound peptide in areaction vial containing a small magnetic bar for gentle stirring. Themixture is flushed with argon, sealed and stirred at room temperaturefor 6 hours. The support-bound peptide is transferred to a filter funneland subject to a series of washes: (i) the first wash is with a 30 ml ofa 0.5% (w/w) solution of sodium diethyldithiocarbonate in DMF; (ii) thesecond wash is with DCM alone; (iii) the third wash is with a 1/1 (v/v)mixture of DCM/DMF; and (iv) the fourth wash is with DMF alone. Apositive Kaiser test indicated the deprotection of the amino sidechained of the Lys⁵⁶.

Lactam Formation:

Activating Agent: 7-azabenztriazol-1-yloxytris (pyrrolindino)phosphonium-hexafluorophosphate (PyAOP) (PerSeptive Biosys. GmbH,GEN076531) is used at a concentration that is 1.4-fold over the 0.105mmol peptide sample size (e.g., 0.105 mmol×1.4 fold×521.7 MW=76.6 mgPyAOP); and NMM is used at a concentration that is 1.5-fold over thePyAOP (e.g., 0.105 mmol×1.4 fold×1.5 fold=0.23 mmol NMM;volume=0.23/0.9M NMM=263 μl).

The lactamization is a cyclization reaction that is carried out with thesupport-bound peptide in an amino acid vial at room temperatureovernight (e.g., ˜16 hours) with gentle agitation. The support-boundpeptide is poured back into the column, washed with DMF, and thenallowed to continue through completion of the cyclization process,wherein a cyclic amide bridge is thereby introduced into the peptide. Anegative Kaiser test is used to indicate the completion of thecyclization process.

Removal of the Final Product from the Support

The support-bound peptide is removed from the synthesizer, placed in amedium filter funnel, washed with DCM to replace the non-volatile DMF,and thoroughly dried under high vacuum for at least two hours, orpreferably, overnight.

Cleavage Mixture (reagent K): 100 ml of a trifluoroacetic acid

(TFA)/Phenol/Water/Thio-Anisol/EDT (82/5/5/5/2.5)(v/v) mixture isprepared. The support-bound peptide (0.5 g) is poured into 7.5 ml ofreagent K with gentle agitation on a rocker, allowed to react for 4hours at room temperature, filtered, and washed with neat TFA. The 7.5ml of reagent K contains the following:

TFA  6.15 ml (Halocarbon) Phenol 0.375 ml (Aldrich) Water 0.375 ml(MillQ) Thio-Anisol 0.375 ml (Aldrich) EDT 0.187 ml (Aldrich) Total  7.5mlPrecipitation of the Peptide

The cleaved (free) peptide solution is filtered through a filter funnelinto a 50 ml round bottom flask. The support is rinsed twice with 4 mlTFA to release the free peptide. The solution of TFA and peptide isconcentrated on a rotavap and added drop wise into cold diethyl etherpreviously treated with activated neutral aluminum oxide to make it freeof peroxide. An excess of ether is used at approximately 10-fold theweight of the support. The support beads from which the peptide wascleaved were stored until the yield was determined and the peptide wascharacterized. The precipitate is collected at room temperature in ascrew-capped 50 ml polypropylene vial after centrifugation for 4 minutesat 2000 rpm in a bench-top centrifuge. The pellets of free peptide werewashed 3× with cold ether, centrifuged and dried under a flow of argon.The precipitate was dissolved in 20% acetonitrile with 0.1% TFA andlyophilized.

Crude Product Characterization

The product is purified and characterized using an analytical HPLCprocedure. A Vydac 218TP54 column (C18 reversed-phase, 4.6 mm×150 mminner column dimensions, and 5 μm particle size). A multisolvent mobilephase is used, and the eluants are a 0.1% TFA/H₂O (solvent A) and a 0.1%TFA/acetonitrile (solvent B).

Elution Conditions: A multisolvent delivery system is used and combinessolvent A and solvent B to alter the polarity of the mobile phase duringelution. The mobile phase is delivered at a flow rate of 1.0 ml/min andat a concentration of 20-50% B for 40 minutes, at a concentration of60-90% B for 5 minutes; at a concentration of 90-20% B for 5 minutes;and at a concentration of 20% B for 10 minutes. The detector is set at214 nm to read 0.5 absorbance units over a full scale.

Sample Preparation:

An aliquot of the product is weighed and dissolved in a mixture of 20%acetonitrile/0.1% TFA (v/v) at a concentration of 2 mg/ml. The solutionis microfuged and 20 μl is injected into the HPLC column. Samplescorresponding to the main and major peaks are collected, SpeedVac dried,and characterized by molecular weights using mass spectroscopy.

Table 1 lists some SDF-1 mimetics that have been prepared by the solidphase peptide synthesis. The underline residues represent a cyclicportion of the SDF-1 mimetic.

TABLE 1 a3) R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Gly-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:3) a4)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Lys-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:4) a5)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro- Phe-Arg-Phe-Phe-GlyGly-Lys-Gly-Leu-Lys-Trp- Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:5) a6) R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Gly-Gly-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:6) a7)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Lys-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:7) a8)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Lys-Lys-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:8) a9)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Gly-Lys-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:9) a10)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Gly-Gly-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:10) a11)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Gly-Lys-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:11) a12)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Lys-Gly-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:12) a13)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Lys-Lys-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:13) a14)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Lys-Lys-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:14) a15)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Gly-Lys-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:15) a16)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Lys-Gly-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:16) a17)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Lys-Lys-Lys-Lys-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:17) a18)R_(N)-Lys-Pro-VaI-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Gly-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:18) a19)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Arg-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-AIa-Leu-Asn-R_(C) (SEQ ID NO:19) a20)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro- Phe-Arg-Phe-Phe-GlyGly-Arg-Gly-Leu-Lys-Trp- Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:20) a21) R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Gly-Gly-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:21) a22)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Arg-Gly-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:22) a23)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Arg-Arg-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:23) a24)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Gly-Arg-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:24) a25)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Gly-Gly-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:25) a26)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Gly-Arg-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:26) a27)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Arg-Gly-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:27) a28)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Arg-Arg-Gly-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:28) a29)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Gly-Arg-Arg-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:29) a30)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Gly-Arg-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:30) a31)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Arg-Gly-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:31) a32)R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-Arg-Arg-Arg-Arg-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C) (SEQ ID NO:32)

Example 2

The efficacy of the SDF-1 mimetics of the invention to bind to mammaliancells and compete with SDF-1 was measured. The experiments includecontacting an SDF-1 mimetic with a cell, and the experiments wereperformed using a human lymphoid cell line of SUP-T1 cells (AmericanType Culture Collection or ATCC) at a concentration of 5×10⁶ cells/ml. ADURAPORE membrane and Millipore MultiScreen 96-well plates were used inthe binding assay, and the membrane was blocked with a PVP/Tween-basedblocking buffer before use. An RPMI-based binding buffer, 0-400 riNM ofSDF-1 or 0-400 μM of an SDF-1 mimetic, a competitive dose of 0.02 nM¹²⁵I-SDF-1 (Amersham), and SUP-T1 cells were added to the wells. Thecells were incubated at 4° C. with shaking for 2 h, followed bytriplicate washes with PBS. Bound ¹²⁵I-SDF-1 was counted using aCliniGamma gamma counter (LKB Wallac).

Experiments were performed in triplicate. Competition curves were fittedwith Graphpad Prism v4.0 after subtracting non-specific binding to bothfilters and cells. The results are expressed as Ki values for thedifferent SDF-1 mimetics and are shown in Table 2, where Ki is thebinding affinity constant, SEM is the standard error of the measurement,and n is the number of samples.

TABLE 2 Compound K_(i) (μM) +/− SEM n Natural SDF-1 0.009 2.379 6 SEQ IDNO: 1 SEQ ID NO: 3 0.663 0.446 4 SEQ ID NO: 4 0.586 0.224 3 SEQ ID NO: 50.378 0.048 3 SEQ ID NO: 6 0.306 0.022 3 SEQ ID NO: 7 0.412 0.245 3 SEQID NO: 8 0.137 0.006 3 SEQ ID NO: 9 0.343 0.252 3 SEQ ID NO: 10 0.4930.097 3 SEQ ID NO: 11 1.213 0.510 3 SEQ ID NO: 12 0.877 0.568 3 SEQ IDNO: 13 2.553 1.288 4 SEQ ID NO: 14 1.173 0.645 4 SEQ ID NO: 15 2.0020.654 4 SEQ ID NO: 16 2.115 1.074 4 SEQ ID NO: 17 1.243 0.517 4 SEQ IDNO: 18 2.308 0.056 4 SEQ ID NO: 19 1.761 0.137 4 SEQ ID NO: 20 3.3510.992 3 SEQ ID NO: 21 2.453 0.561 4 SEQ ID NO: 22 0.744 0.143 4 SEQ IDNO: 23 1.675 0.478 4 SEQ ID NO: 24 1.780 0.921 4 SEQ ID NO: 25 1.0780.243 4 SEQ ID NO: 26 1.265 0.730 4 SEQ ID NO: 27 1.535 0.673 4 SEQ IDNO: 28 0.741 0.360 4 SEQ ID NO: 29 1.261 0.462 4 SEQ ID NO: 30 1.1120.323 4 SEQ ID NO: 31 0.797 0.240 4 SEQ ID NO: 32 0.833 0.268 4

Example 3

The efficacy of the chemokine analogs of the invention to activatemammalian cell receptors is demonstrated by their ability to mobilizeintracellular calcium in SUP-T1 cells. The experiments includecontacting an SDF-1 mimetic with a cell. For the experiments, SUP-T1cells (ATCC) were plated on the day of the experiment using 1.2×10⁵cells per well in 96-well black-wall/clear-bottom plates coated withpoly-D-lysine (BD Biosciences) and loaded using a fluorescent calciumindicator. The indicator used was from a FLIPR Calcium 3 assay kit,component A, (Molecular Probes) and was loaded in the cell for 1 hr at37° C. The intracellular calcium mobilization in response to theappropriate analogue was measured at 37° C. by monitoring thefluorescence as a function of time simultaneously in all the wells usinga Flexstation Fluorometric Imaging Plate Reader (Molecular Devices). TheEC₅₀ values of the different SDF-1 mimetics of the present invention aresummarized in Table 3.

TABLE 3 Compound EC₅₀ (μM) SEQ ID NO: 3 0.346 SEQ ID NO: 4 0.312 SEQ IDNO: 5 0.211 SEQ ID NO: 6 0.283 SEQ ID NO: 7 0.281 SEQ ID NO: 8 0.304 SEQID NO: 9 0.225 SEQ ID NO: 10 0.233 SEQ ID NO: 11 0.228 SEQ ID NO: 120.307 SEQ ID NO: 13 0.137 SEQ ID NO: 14 0.092 SEQ ID NO: 15 0.157 SEQ IDNO: 16 0.140 SEQ ID NO: 17 0.316 SEQ ID NO: 18 0.219 SEQ ID NO: 19 0.253SEQ ID NO: 20 0.307 SEQ ID NO: 21 0.361 SEQ ID NO: 22 0.171 SEQ ID NO:23 0.202 SEQ ID NO: 24 0.173 SEQ ID NO: 25 0.132 SEQ ID NO: 26 0.248 SEQID NO: 27 4.315 SEQ ID NO: 28 0.597 SEQ ID NO: 29 1.873 SEQ ID NO: 300.178 SEQ ID NO: 31 0.709 SEQ ID NO: 32 1.117

Example 4

The ability of the SDF-1 mimetics to induce the survival of HumanUmbilical Vein Endothelial Cells (HUVEC) in a serum free medium isdemonstrated using the MTT assay to analyse cell viability after peptidetreatment. An MTT assay is a standard colorimetric assay for measuringcell growth. The amount of yellow MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) thatoxidizes to purple formazan is measured with a spectrometer. Theoxidation occurs only when mitochondrial reductase enzymes are activeand is directly related to the number of viable cells. The production ofpurple formazan in cells treated with an agent is measured relative tothe production in control cells.

The experiments include contacting an SDF-1 mimetic with a cell. TheHUVECs were cultured using an endothelial growth medium containing 10%fetal bovine serum (FBS) until they were 80% confluent in a humid, 5%CO₂, 37° C. incubator. Cells were trypsinized, counted by hemocytometer,and seeded for an MTT survival assay. Compounds and controls wereprepared in a serum-free culture medium and incubated for 72 hours. Tomeasure cell survival, an MTT survival assay was performed as follows:10,000 HUVEC's in 100 μL of culture medium were plated into each well ofa 96-well plate for 48 hours. Cells were washed and treated withdifferent concentrations of a compound for 72 hours. 10 μL MTT (5 mg/mL)was added for 4 hours, the colored precipitate was dissolved usingacidic (1N HCl) isopropanol, and the absorbance was measured at awavelength of 570-650 nm. FIGS. 1-5 show the results of MTT calorimetricassays that demonstrate the viability of human umbilical veinendothelial cells after treatment of the cells with SEQ ID NOs:3, 5, 9,13, and 24 according to some embodiments of the present invention. Theresults are expressed as percentage of the effect of recombinant humanSDF-1 used at 125 μM.

Example 5

The ability of the peptides of this invention to induce thedifferentiation of Human Vein Endothelial Cells is demonstrated with thematrigel tube formation assay. For these experiments, HUVECs werecultured using endothelial growth medium containing 10% FBS until theywere 80% confluent in a humid, 5% CO₂, 37° C. incubator. Cells weretrypsinized, counted by hemocytometer, and seeded for MATRIGEL TubeFormation Assays. Compounds and controls were prepared in MATRIGEL andincubated for 72 hours.

The experiments include contacting an SDF-1 mimetic with a cell. TheMATRIGEL Tube Formation Assay was performed as follows: 8,000 cells in50 μL were plated into each well of a 96-well plate pre-coated with 150μL Growth Factor Reduced MATRIGEL and varying concentrations ofcompounds. FIGS. 6 a through 6 c show that SDF-1 mimetics inducedifferentiation of human umbilical vein endothelial cells as seen by theformation of tube structures when tested using a MATRIGEL tube formationassay according to some embodiments of the present invention. FIG. 6 aillustrates a control sample having no SDF-1 mimetic. FIG. 6 billustrates a sample having SEQ ID NO:13 at a concentration of 5 μg/ml,and FIG. 6 c illustrates a sample having SEQ ID NO:24 also at aconcentration of 5 μg/ml. Accordingly, these results show thatendothelial tube formation was strongly induced through theadministration of SEQ ID NOs: 13 and 24.

Example 6

The SDF-1 mimetics of the present invention are capable of inducingneo-vessel formation as demonstrated in an aortic ring assay. Theexperiments include contacting an SDF-1 mimetic with a cell. FIGS. 7 a-7c show that SDF-1 mimetics induce neo-vascularization when tested usinga rat aortic ring assay according to some embodiments of the presentinvention. For the experiments, aorta were dissected from 6 month oldfemale Sprague Dawley rats and cut into 1 mm cross sections to createaortic rings. The aortic rings were embedded in collagen contained andcovered in a serum-free medium for 7 days. FIG. 7 a illustrates acontrol sample having no SDF-1 mimetic. FIG. 7 b illustrates a samplehaving SEQ ID NO: 13 at a concentration of 5 μg/ml, and FIG. 7 cillustrates a sample having SEQ ID NO:24 also at a concentration of 5μg/ml. Accordingly, these results show that SEQ ID NOs:13 and 24 induceneo-vessel formation.

Example 7

Blood vessel growth is necessary for normal tissue homeostatis. Methodsto measure neovascularization are useful in testing compounds for theireffect on angiogenesis and their ability to induce a vascular supply topromote wound healing. A MATRIGEL plug assay is an extract of basementmembrane proteins that reconstitute into a gel when injectedsubcutaneously into a mammal. This gel can support an intense vascularresponse when supplemented with compounds capable of creating thevascular response, such as the SDF-1 mimetics of the present invention.

The experiments include contacting an SDF-1 mimetic with a cell. FIGS. 8a through 8 d show that SDF-1 mimetics induce blood vessel formation invivo when tested using a MATRIGEL plug assay according to someembodiments of the present invention. In this example, SEQ ID NO:13 wastested in mice by incorporating the mimetic into a MATRIGEL andinjecting the supplemented MATRIGEL into the mice using the MATRIGELplug assay procedure. Human and mouse SDF-1 are approximately 92%identical.

The mice were euthanized, and the gel was removed and stained withhematoxylin and eosin (H+E). FIG. 8 a illustrates a control samplehaving no SDF-1 mimetic for a three week period. FIG. 8 b through 8 dillustrate samples having SEQ ID NO:13 at a concentration of 1 μM, 5 μM,and 10 μM, respectively, for the three week period. Accordingly, theseresults show that SEQ ID NO:13 induces blood vessel formation in vivo.

Example 8

Agents can be attached as modifying groups that are pendant or in-chainwith an SDF-1 mimetic. A trifunctional amino acid, for example, can beincorporated into the SDF-1 mimetic as a linker and the thirdfunctionality can be connected to an agent. Protecting groups can beused to selectively attach an agent to the trifunctional amino acid.Benzyl esters are one type of protecting group that can be used for alysine carboxyl, for example, and t-butoxycarbonyl can be used for aminogroups such as, for example, the amino group in glutamic acid.

Amino, hydroxyl and carboxyl groups can be used, for example, as aconnecting site for agents. Carboxyl groups can be used as a connectingsite for agents having, for example, amino, hydroxyl, or thiol groups.Coupling agents include, but are not limited to,1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and1,3-dicyclohexylcarbodiimide (DCC).

An example of an amine functional compound is 4-amino-TEMPO, anantioxidant and antihypertensive that can be administered as a codrug incombination with an SDF-1 mimetic. Such an amine functional compound maybe connected to an amino acid sequence containing free carboxyls suchas, for example, the lysine-derived carboxyls, by first activating thecarboxyls and coupling the amine in a solvent under agitation. Thecarboxyls may be activated with, for example, N-hydroxysuccinimide (NHS)and DCC in a solvent such as, for example, THF or chloroform, whichproduces N-hydroxysuccinimidyl ester. Examples of the solvent that maybe used to couple the amine to the carboxyls include, but are notlimited to, THF and DMF. One of skill will appreciate that otherlinkages can be preselected and created in order to increase the rate ofrelease of a desired agent from an SDF-1 mimetic such as, for example,an ester or an anhydride linkage.

In some embodiments, the reaction can occur at a temperature rangingfrom about 5° C. to about 50° C., from about 15° C. to about 35° C.,from about 20° C. to about 30° C., or any range therein. In someembodiments, the reaction time can range from about 0.5 hours to about24 hours, from about 1 hour to about 18 hours, from about 4 hours toabout 16 hours, from about 6 hours to about 12 hours, or any rangetherein.

A benzyl ester protecting group can be removed from a lysine carboxyl byhydrogenolysis with hydrogen gas over a catalyst such as, for example,palladium or platinum on carbon. Examples of suitable solvents include,but are not limited to, ethanol, methanol, isopropanol, and THF. Thereaction may be conducted under about 1 atm of hydrogen for about 6hours to about 24 hours, for about 8 hours to about 16 hours, for about10 hours to about 14 hours, or any range therein.

Example 9

A glycosaminoglycan can be connected to an amine functional group as analdehyde-terminated heparin, for example, to provide additional controlover the behavior of the SDF-1 mimetic in vivo and/or to provide acodrug form of the mimetic. An example of an aldehyde-terminated heparinis represented by the following formula:

wherein p is an integer not equal to 0.

The aldehyde-terminated heparin can be combined with the aminefunctional group in a DMF/water solvent and subsequently reduced withNaCNBH₃ to produce a heparin linked to an SDF-1 mimetic through an amidebond.

Example 10

The behavior of an SDF-1 mimetic can also be modified by attaching it toother compounds as well. The mimetic can be modified, for example, witha polyalkylene glycol, such as poly(ethylene glycol) (PEG) using avariety of techniques known to one of skill in the art. There are avariety of available PEG sizes and derivatives that are commerciallydesigned for specific applications such as, for example, attachment to avariety of different chemical functionalities including, but not limitedto, amines, thiols, hydroxyls, sulfhydryls, and carboxyls.

In one example, an amine group of an SDF-1 mimetic can be combined witha carboxyl-terminated PEG (Nektar Corp.) in the presence of, forexample, EDC or DCC to form a pegylated structure through formation ofan amide bond between the SDF-1 mimetic and the PEG.

In another example, either a succinimidyl derivative of MPEG (NektarCorp.) or an isocyanate-terminated MPEG (Nektar Corp.) can be combinedwith an SDF-1 mimetic under conditions known to those of skill in theart. In another example, the carboxyl group of an SDF-1 mimetic can beactivated with, for example, EDC or DCC and combined with anamino-terminated mPEG (Nektar Corp.) In another example, an amine groupof an SDF-1 mimetic can be combined with a methacrylate-terminated MPEG(Nektar Corp.) in the presence of an initiator capable of undergoingthermal or photolytic free radical decomposition. Examples of suitableinitiators include benzyl-N,N-diethyldithiocarbamate orp-xylene-N,N-diethyldithiocarbamate.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, that there are many equivalents tothe specific embodiments described herein that have been described andenabled to the extent that one of skill in the art can practice theinvention well-beyond the scope of the specific embodiments taughtherein. Such equivalents are intended to be encompassed by the followingclaims. In addition, there are numerous lists and Markush groups taughtand claimed herein. One of skill will appreciate that each such list andgroup contains various species and can be modified by the removal, oraddition, of one or more of species, since every list and group taughtand claimed herein may not be applicable to every embodiment feasible inthe practice of the invention. All publications, patents, and patentapplications mentioned in this application are herein incorporated byreference into the specification to the same extent as if each wasspecifically indicated to be herein incorporated by reference in itsentirety.

1. A method of inducing neo-vessel formation comprising contacting anendothelial cell with a composition comprising an SDF-1 mimetic having astructure selected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(c)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(c) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; and wherein, the linkercomprises at least one amino acid having a side chain bearing a positivecharge and is not tetralysine.
 2. The method of claim 1, wherein Xaa₁,Xaa₂, Xaa₃, and Xaa₄ are each independently selected from a groupconsisting of Gly, L- or D-Lys, L- or D-Arg, L- or D-His, and L- orD-Orn.
 3. The method of claim 1, wherein the SDF-1 mimetic comprises anamino acid sequence selected from a group consisting of SEQ ID NO:3 toSEQ ID NO:16.
 4. The method of claim 1, wherein the SDF-1 mimeticcomprises an amino acid sequence selected from a group consisting of SEQID NO:18 to SEQ ID NO:32.
 5. The method of claim 1, wherein R_(L)consists of an alkylene having a backbone consisting of 1 to 14 carbonatoms.
 6. The method of claim 1, wherein R_(N) or R_(C) comprises apoly(ethylene glycol) or a derivative thereof having a molecular weightof less than 20,000 Daltons.
 7. The method of claim 1, wherein theinducing of neo-vessel formation includes mobilizing endothelial cells,mobilizing endothelial progenitor cells, mobilizing hematopoietic stemcells, inducing differentiation of endothelial cells, inducing survivalof endothelial cells, inducing retention of endothelial cells, inducingendothelial tube formation, or any combination thereof.
 8. The method ofclaim 1 further comprising contacting the SDF-1 mimetic with anendothelial progenitor cell, a hematopoietic stem cell, or a combinationthereof.
 9. A method of inducing angiogenesis in a tissue of a subjectcomprising administering to a subject an effective amount of acomposition comprising an SDF-1 mimetic having a structure selected fromthe group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; and wherein, the linkercomprises at least one amino acid having a side chain bearing a positivecharge and is not tetralysine.
 10. The method of claim 9, wherein theinducing angiogenesis includes mobilizing endothelial cells, mobilizingendothelial progenitor cells, mobilizing hematopoietic stem cells,inducing differentiation of endothelial cells, inducing survival ofendothelial cells, inducing retention of endothelial cells, inducingendothelial tube formation, or any combination thereof.
 11. The methodof claim 9, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ are each independentlyselected from a group consisting of Gly, L- or D-Lys, L- or D-Arg, L- orD-His, and L- or D-Orn.
 12. The method of claim 9, wherein R_(N) orR_(C) comprises a poly(ethylene glycol) or a derivative thereof having amolecular weight of less than 20,000 Daltons.
 13. The method of claim 9,wherein the SDF-1 mimetic comprises an amino acid sequence selected froma group consisting of SEQ ID NO:3 to SEQ ID NO:16.
 14. The method ofclaim 9, wherein the SDF-1 mimetic comprises an amino acid sequenceselected from a group consisting of SEQ ID NO:18 to SEQ ID NO:32. 15.The method of claim 9, wherein R_(L) consists of an alkylene having abackbone consisting of 1 to 14 carbon atoms.
 16. The method of claim 9,wherein the angiogenesis is induced to prevent or repair injury to bloodvessels and to maintain or increase blood supply to the tissue.
 17. Themethod of claim 9, wherein the angiogenesis is induced to prevent,treat, or ameliorate the symptoms of, a disease comprising ischemia,cardiovascular disease, peripheral vascular disease, a renal disease,diabetes, pulmonary dysfunction, or a microvascular angiopathy.
 18. Themethod of claim 9, wherein the angiogenesis is induced to treat, orameliorate the symptoms of, a cancer by creating a vascularity within atumor for transport of an agent into the tumor.
 19. The method of claim9 further comprising transplanting endothelial progenitor cells into thetissue.
 20. The method of claim 19, wherein the transplanting comprisesadministering an effective amount of an agent in a subject to stimulatethe subject's bone marrow to mobilize the endothelial progenitor cellsfor the transplanting.
 21. The method of claim 20, wherein the agentcomprises the SDF-1 mimetic.
 22. The method of claim 9 furthercomprising administering a poly(ethylene glycol) or copolymers orderivatives thereof, a glycosaminoglycan, heparin or derivativesthereof, hirudin, a phospholipid, phosphatidylcholine, or combinationsthereof.
 23. An article of manufacture useful in practicing the methodof claim 9 comprising: (a) a first composition comprising an SDF-1mimetic having a structure selected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, abiochemical label, and an N-terminal modifier capable of reducing theability of the SDF-1 mimetic to act as a substrate for aminopeptidases;R_(C) is selected from a group consisting of a hydroxyl group,poly(ethylene glycol) or a derivative thereof, a biochemical label, anda C-terminal modifier capable of reducing the ability of the SDF-1mimetic to act as a substrate for carboxypeptidases; and, the linkerconsists of four amino acids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂,Xaa₃, and Xaa₄ are each independently selected from a group consistingof (a) any natural amino acid, provided that the natural amino acid isnot L- or D-Cys, and (b) any non-natural amino acid having the followingstructure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; and wherein, the linkercomprises at least one amino acid having a side chain bearing a positivecharge and is not tetralysine; and, (b) instructions for administeringthe first composition to a subject and monitoring the subject.
 24. Thearticle of manufacture of claim 23, wherein the SDF-1 mimetic comprisesan amino acid sequence selected from a group consisting of SEQ ID NO:3to SEQ ID NO:16.
 25. The article of manufacture of claim 23, wherein theSDF-1 mimetic comprises an amino acid sequence selected from a groupconsisting of SEQ ID NO:18 to SEQ ID NO:32.
 26. The article ofmanufacture of claim 23, wherein the first composition comprises asecond agent.
 27. The article of manufacture of claim 26, wherein thesecond agent is in a codrug form with the SDF-1 mimetic.
 28. The articleof manufacture of claim 23, wherein the first composition comprises asecond agent selected from a group consisting of poly(ethylene glycol)or derivatives thereof, a glycosaminoglycan, heparin or derivativesthereof, a phospholipid, and phosphatidylcholine.
 29. The article ofmanufacture of claim 23 further comprising a second compositioncomprising a second agent for administering in combination with thefirst composition, wherein the instructions further compriseinstructions for administering the second composition and monitoring thepatient.
 30. The article of manufacture of claim 29, wherein the secondcomposition comprises a second agent selected from a group consisting ofantiproliferatives, antineoplastics, and antimitotics.
 31. The articleof manufacture of claim 23 further comprising instructions forstimulating bone marrow to mobilize endothelial progenitor cells andtransplanting the endothelial progenitor cells into a subject to induceangiogenesis.
 32. The article of manufacture of claim 23 furthercomprising instructions for mobilizing and retaining hematopoietic stemcells close to angiogenic vessels to induce angiogenesis.
 33. Acomposition comprising an SDF-1 mimetic selected from the groupconsisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; and wherein, the linkercomprises at least one amino acid having a side chain bearing a positivecharge and is not tetralysine.
 34. The composition of claim 33, whereinXaa₁, Xaa₂, Xaa₃, and Xaa₄ are each independently selected from a groupconsisting of Gly, L- or D-Lys, L- or D-Arg, L- or D-His, and L- orD-Orn.
 35. The composition of claim 33, wherein the SDF-1 mimeticcomprises an amino acid sequence selected from a group consisting of SEQID NO:3 to SEQ ID NO:16.
 36. The composition of claim 33, wherein theSDF-1 mimetic comprises an amino acid sequence selected from a groupconsisting of SEQ ID NO:18 to SEQ ID NO:32.
 37. The composition of claim33, wherein R_(L) consists of an alkylene having a backbone consistingof 1 to 14 carbon atoms.
 38. The composition of claim 33, wherein R_(N)or R_(C) comprises a poly(ethylene glycol) or a derivative thereofhaving a molecular weight of less than 20,000 Daltons.
 39. A method ofinducing neo-vessel formation comprising contacting an endothelial cellwith a composition comprising an SDF-1 mimetic having a structureselected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; wherein at least one ofXaa₁, Xaa₂, Xaa₃, and Xaa₄ is a non-natural amino acid, wherein R_(L)consists of an alkylene having a backbone consisting of 1 to 14 carbonatoms; and wherein, the linker comprises at least one amino acid havinga side chain bearing a positive charge.
 40. A method of inducingangiogenesis in a tissue of a subject comprising administering to asubject an effective amount of a composition comprising an SDF-1 mimetichaving a structure selected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; wherein at least one ofXaa₁, Xaa₂, Xaa₃, and Xaa₄ is a non-natural amino acid, wherein R_(L)consists of an alkylene having a backbone consisting of 1 to 14 carbonatoms; and wherein, the linker comprises at least one amino acid havinga side chain bearing a positive charge.
 41. A method of inducingangiogenesis in a tissue of a subject comprising administering to asubject an effective amount of a composition comprising an SDF-1 mimetichaving a structure selected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; and wherein, the linkercomprises at least one amino acid having a side chain bearing a positivecharge; and, the angiogenesis is induced to treat, or ameliorate thesymptoms of, a cancer by creating a vascularity within a tumor fortransport of an agent into the tumor.
 42. A composition comprising anSDF-1 mimetic selected from the group consisting of:R_(N)-Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Ala-Pro-Phe-Arg-Phe-Phe-[linker]-Leu-Lys-Trp-Ile-Gln-Glu-Tyr-Leu-Glu-Lys-Ala-Leu-Asn-R_(C)(SEQ ID NO:2) and conservatively modified variants, prodrugs, andcodrugs thereof; wherein, the underlined residues Lys-Trp-Ile-Gln-Gluform a cyclic portion of the mimetic; R_(N) is selected from a groupconsisting of hydrogen, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and an N-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for aminopeptidases; R_(C) is selected from a group consistingof a hydroxyl group, poly(ethylene glycol) or a derivative thereof, aglycosaminoglycan, a biochemical label, and a C-terminal modifiercapable of reducing the ability of the SDF-1 mimetic to act as asubstrate for carboxypeptidases; and, the linker consists of four aminoacids, -Xaa₁-Xaa₂-Xaa₃-Xaa₄-, wherein Xaa₁, Xaa₂, Xaa₃, and Xaa₄ areeach independently selected from a group consisting of (a) any naturalamino acid, provided that the natural amino acid is not L- or D-Cys, and(b) any non-natural amino acid having the following structure:

wherein, R_(L) is selected from a group consisting of saturated andunsaturated aliphatics and heteroaliphatics consisting of 20 or fewercarbon atoms that are optionally substituted with (i) a hydroxyl,carboxyl, amino, amido, or imino group; (ii) an aromatic group havingfrom 5 to 7 members in the ring; or (iii) a group having from 0 to 10carbon atoms and bearing a positive charge; wherein at least one ofXaa₁, Xaa₂, Xaa₃, and Xaa₄ is a non-natural amino acid, wherein R_(L)consists of an alkylene having a backbone consisting of 1 to 14 carbonatoms; and wherein, the linker comprises at least one amino acid havinga side chain bearing a positive charge.
 43. A pharmaceutical compositioncomprising the composition of claim
 33. 44. A pharmaceutical compositioncomprising the composition of claim 42.